WO2023074748A1 - 抗ウイルスアンチセンスオリゴヌクレオチド - Google Patents
抗ウイルスアンチセンスオリゴヌクレオチド Download PDFInfo
- Publication number
- WO2023074748A1 WO2023074748A1 PCT/JP2022/039960 JP2022039960W WO2023074748A1 WO 2023074748 A1 WO2023074748 A1 WO 2023074748A1 JP 2022039960 W JP2022039960 W JP 2022039960W WO 2023074748 A1 WO2023074748 A1 WO 2023074748A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- region
- pharmaceutically acceptable
- acceptable salt
- seq
- cov
- Prior art date
Links
- 239000000074 antisense oligonucleotide Substances 0.000 title claims abstract description 109
- 238000012230 antisense oligonucleotides Methods 0.000 title claims abstract description 109
- 108091034117 Oligonucleotide Proteins 0.000 title claims abstract description 75
- 230000000840 anti-viral effect Effects 0.000 title claims description 12
- 150000003839 salts Chemical class 0.000 claims abstract description 68
- 241001678559 COVID-19 virus Species 0.000 claims abstract description 55
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 21
- 125000003729 nucleotide group Chemical group 0.000 claims description 101
- 239000002773 nucleotide Substances 0.000 claims description 97
- 239000002777 nucleoside Substances 0.000 claims description 49
- 125000003835 nucleoside group Chemical group 0.000 claims description 47
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 36
- 108020004707 nucleic acids Proteins 0.000 claims description 36
- 102000039446 nucleic acids Human genes 0.000 claims description 36
- 150000007523 nucleic acids Chemical class 0.000 claims description 35
- 108060004795 Methyltransferase Proteins 0.000 claims description 32
- 241000700605 Viruses Species 0.000 claims description 26
- 241000315672 SARS coronavirus Species 0.000 claims description 22
- 230000000295 complement effect Effects 0.000 claims description 22
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 claims description 14
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 11
- 230000009385 viral infection Effects 0.000 claims description 11
- 101800001631 3C-like serine proteinase Proteins 0.000 claims description 10
- 102000016397 Methyltransferase Human genes 0.000 claims description 10
- 101800000515 Non-structural protein 3 Proteins 0.000 claims description 10
- 101800004803 Papain-like protease Proteins 0.000 claims description 10
- 101800002227 Papain-like protease nsp3 Proteins 0.000 claims description 10
- 101800001074 Papain-like proteinase Proteins 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 108060002716 Exonuclease Proteins 0.000 claims description 9
- 101800000509 Non-structural protein 8 Proteins 0.000 claims description 9
- 208000036142 Viral infection Diseases 0.000 claims description 9
- 102000013165 exonuclease Human genes 0.000 claims description 9
- 230000002401 inhibitory effect Effects 0.000 claims description 9
- 101800000535 3C-like proteinase Proteins 0.000 claims description 8
- 101800000120 Host translation inhibitor nsp1 Proteins 0.000 claims description 8
- 101800000512 Non-structural protein 1 Proteins 0.000 claims description 8
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 claims description 8
- 108020005345 3' Untranslated Regions Proteins 0.000 claims description 7
- 101000748061 Acholeplasma phage L2 Uncharacterized 16.1 kDa protein Proteins 0.000 claims description 7
- 101000947615 Clostridium perfringens Uncharacterized 38.4 kDa protein Proteins 0.000 claims description 7
- 101000964391 Enterococcus faecalis UPF0145 protein Proteins 0.000 claims description 7
- 101000748063 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 11.1 kDa protein in rep-hol intergenic region Proteins 0.000 claims description 7
- 101000790840 Klebsiella pneumoniae Uncharacterized 49.5 kDa protein in cps region Proteins 0.000 claims description 7
- 101000596375 Severe acute respiratory syndrome coronavirus 2 ORF7b protein Proteins 0.000 claims description 7
- 150000004677 hydrates Chemical class 0.000 claims description 7
- 108020003589 5' Untranslated Regions Proteins 0.000 claims description 5
- 150000004713 phosphodiesters Chemical class 0.000 claims description 5
- 101800000933 Non-structural protein 10 Proteins 0.000 claims description 4
- 101800000514 Non-structural protein 4 Proteins 0.000 claims description 4
- 101800000507 Non-structural protein 6 Proteins 0.000 claims description 4
- 101800000510 Non-structural protein 7 Proteins 0.000 claims description 4
- 101800000482 Non-structural protein 9 Proteins 0.000 claims description 4
- 101800001554 RNA-directed RNA polymerase Proteins 0.000 claims description 4
- 101000779242 Severe acute respiratory syndrome coronavirus 2 ORF3a protein Proteins 0.000 claims description 4
- 230000000692 anti-sense effect Effects 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 2
- 230000036571 hydration Effects 0.000 claims 1
- 238000006703 hydration reaction Methods 0.000 claims 1
- 230000008685 targeting Effects 0.000 abstract description 3
- 239000002585 base Substances 0.000 description 89
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 37
- -1 DIG nucleic acid Chemical class 0.000 description 20
- 230000000694 effects Effects 0.000 description 14
- 239000000203 mixture Substances 0.000 description 14
- 238000012360 testing method Methods 0.000 description 14
- 239000000126 substance Substances 0.000 description 13
- 239000013613 expression plasmid Substances 0.000 description 11
- 238000012986 modification Methods 0.000 description 10
- 230000004048 modification Effects 0.000 description 10
- 239000013642 negative control Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 241000008910 Severe acute respiratory syndrome-related coronavirus Species 0.000 description 7
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Natural products O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 7
- 239000002552 dosage form Substances 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 241000711573 Coronaviridae Species 0.000 description 6
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 6
- 125000000217 alkyl group Chemical group 0.000 description 6
- 125000004432 carbon atom Chemical group C* 0.000 description 6
- 239000012228 culture supernatant Substances 0.000 description 6
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 6
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 5
- 125000003545 alkoxy group Chemical group 0.000 description 5
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 5
- 238000002869 basic local alignment search tool Methods 0.000 description 5
- 239000000969 carrier Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000004520 electroporation Methods 0.000 description 5
- 230000002458 infectious effect Effects 0.000 description 5
- 239000002502 liposome Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- 102000006335 Phosphate-Binding Proteins Human genes 0.000 description 4
- 108010058514 Phosphate-Binding Proteins Proteins 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 125000003118 aryl group Chemical group 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 229940104302 cytosine Drugs 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- 229940035893 uracil Drugs 0.000 description 4
- HWPZZUQOWRWFDB-UHFFFAOYSA-N 1-methylcytosine Chemical compound CN1C=CC(N)=NC1=O HWPZZUQOWRWFDB-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000008904 Betacoronavirus Species 0.000 description 3
- 208000025721 COVID-19 Diseases 0.000 description 3
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 108091028664 Ribonucleotide Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 229960000643 adenine Drugs 0.000 description 3
- 125000002947 alkylene group Chemical group 0.000 description 3
- 125000003435 aroyl group Chemical group 0.000 description 3
- 125000002091 cationic group Chemical group 0.000 description 3
- 125000004093 cyano group Chemical group *C#N 0.000 description 3
- 125000000753 cycloalkyl group Chemical group 0.000 description 3
- 239000005547 deoxyribonucleotide Substances 0.000 description 3
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 3
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 3
- 229910052736 halogen Inorganic materials 0.000 description 3
- 150000002367 halogens Chemical class 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 239000012669 liquid formulation Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 239000002336 ribonucleotide Substances 0.000 description 3
- 125000002652 ribonucleotide group Chemical group 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- HPZMWTNATZPBIH-UHFFFAOYSA-N 1-methyladenine Chemical compound CN1C=NC2=NC=NC2=C1N HPZMWTNATZPBIH-UHFFFAOYSA-N 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- FZWGECJQACGGTI-UHFFFAOYSA-N 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one Chemical compound NC1=NC(O)=C2N(C)C=NC2=N1 FZWGECJQACGGTI-UHFFFAOYSA-N 0.000 description 2
- 101800000504 3C-like protease Proteins 0.000 description 2
- OVONXEQGWXGFJD-UHFFFAOYSA-N 4-sulfanylidene-1h-pyrimidin-2-one Chemical compound SC=1C=CNC(=O)N=1 OVONXEQGWXGFJD-UHFFFAOYSA-N 0.000 description 2
- OIVLITBTBDPEFK-UHFFFAOYSA-N 5,6-dihydrouracil Chemical compound O=C1CCNC(=O)N1 OIVLITBTBDPEFK-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 102100035765 Angiotensin-converting enzyme 2 Human genes 0.000 description 2
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000929928 Homo sapiens Angiotensin-converting enzyme 2 Proteins 0.000 description 2
- 101000611202 Homo sapiens Peptidyl-prolyl cis-trans isomerase B Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 102100040283 Peptidyl-prolyl cis-trans isomerase B Human genes 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229920002873 Polyethylenimine Polymers 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108091027544 Subgenomic mRNA Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 108700005078 Synthetic Genes Proteins 0.000 description 2
- 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 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 125000001589 carboacyl group Chemical group 0.000 description 2
- 229920006317 cationic polymer Polymers 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 2
- 229940112141 dry powder inhaler Drugs 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 102000048657 human ACE2 Human genes 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- DRAVOWXCEBXPTN-UHFFFAOYSA-N isoguanine Chemical compound NC1=NC(=O)NC2=C1NC=N2 DRAVOWXCEBXPTN-UHFFFAOYSA-N 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 239000008215 water for injection Substances 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- YIMATHOGWXZHFX-WCTZXXKLSA-N (2r,3r,4r,5r)-5-(hydroxymethyl)-3-(2-methoxyethoxy)oxolane-2,4-diol Chemical compound COCCO[C@H]1[C@H](O)O[C@H](CO)[C@H]1O YIMATHOGWXZHFX-WCTZXXKLSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- FYADHXFMURLYQI-UHFFFAOYSA-N 1,2,4-triazine Chemical compound C1=CN=NC=N1 FYADHXFMURLYQI-UHFFFAOYSA-N 0.000 description 1
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 1
- SATCOUWSAZBIJO-UHFFFAOYSA-N 1-methyladenine Natural products N=C1N(C)C=NC2=C1NC=N2 SATCOUWSAZBIJO-UHFFFAOYSA-N 0.000 description 1
- KIQMCGMHGVXDFW-UHFFFAOYSA-N 1-methylhypoxanthine Chemical compound O=C1N(C)C=NC2=C1NC=N2 KIQMCGMHGVXDFW-UHFFFAOYSA-N 0.000 description 1
- QIJIUJYANDSEKG-UHFFFAOYSA-N 2,4,4-trimethylpentan-2-amine Chemical class CC(C)(C)CC(C)(C)N QIJIUJYANDSEKG-UHFFFAOYSA-N 0.000 description 1
- HLYBTPMYFWWNJN-UHFFFAOYSA-N 2-(2,4-dioxo-1h-pyrimidin-5-yl)-2-hydroxyacetic acid Chemical compound OC(=O)C(O)C1=CNC(=O)NC1=O HLYBTPMYFWWNJN-UHFFFAOYSA-N 0.000 description 1
- SGAKLDIYNFXTCK-UHFFFAOYSA-N 2-[(2,4-dioxo-1h-pyrimidin-5-yl)methylamino]acetic acid Chemical compound OC(=O)CNCC1=CNC(=O)NC1=O SGAKLDIYNFXTCK-UHFFFAOYSA-N 0.000 description 1
- YSAJFXWTVFGPAX-UHFFFAOYSA-N 2-[(2,4-dioxo-1h-pyrimidin-5-yl)oxy]acetic acid Chemical compound OC(=O)COC1=CNC(=O)NC1=O YSAJFXWTVFGPAX-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- NOIRDLRUNWIUMX-UHFFFAOYSA-N 2-amino-3,7-dihydropurin-6-one;6-amino-1h-pyrimidin-2-one Chemical compound NC=1C=CNC(=O)N=1.O=C1NC(N)=NC2=C1NC=N2 NOIRDLRUNWIUMX-UHFFFAOYSA-N 0.000 description 1
- MWBWWFOAEOYUST-UHFFFAOYSA-N 2-aminopurine Chemical compound NC1=NC=C2N=CNC2=N1 MWBWWFOAEOYUST-UHFFFAOYSA-N 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical group OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- XMSMHKMPBNTBOD-UHFFFAOYSA-N 2-dimethylamino-6-hydroxypurine Chemical compound N1C(N(C)C)=NC(=O)C2=C1N=CN2 XMSMHKMPBNTBOD-UHFFFAOYSA-N 0.000 description 1
- SMADWRYCYBUIKH-UHFFFAOYSA-N 2-methyl-7h-purin-6-amine Chemical compound CC1=NC(N)=C2NC=NC2=N1 SMADWRYCYBUIKH-UHFFFAOYSA-N 0.000 description 1
- KOLPWZCZXAMXKS-UHFFFAOYSA-N 3-methylcytosine Chemical compound CN1C(N)=CC=NC1=O KOLPWZCZXAMXKS-UHFFFAOYSA-N 0.000 description 1
- VPLZGVOSFFCKFC-UHFFFAOYSA-N 3-methyluracil Chemical compound CN1C(=O)C=CNC1=O VPLZGVOSFFCKFC-UHFFFAOYSA-N 0.000 description 1
- GJAKJCICANKRFD-UHFFFAOYSA-N 4-acetyl-4-amino-1,3-dihydropyrimidin-2-one Chemical compound CC(=O)C1(N)NC(=O)NC=C1 GJAKJCICANKRFD-UHFFFAOYSA-N 0.000 description 1
- AOZBINSIAHDCQU-UHFFFAOYSA-N 5-(2-oxopropyl)-1h-pyrimidine-2,4-dione Chemical compound CC(=O)CC1=CNC(=O)NC1=O AOZBINSIAHDCQU-UHFFFAOYSA-N 0.000 description 1
- MQJSSLBGAQJNER-UHFFFAOYSA-N 5-(methylaminomethyl)-1h-pyrimidine-2,4-dione Chemical compound CNCC1=CNC(=O)NC1=O MQJSSLBGAQJNER-UHFFFAOYSA-N 0.000 description 1
- WPYRHVXCOQLYLY-UHFFFAOYSA-N 5-[(methoxyamino)methyl]-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CONCC1=CNC(=S)NC1=O WPYRHVXCOQLYLY-UHFFFAOYSA-N 0.000 description 1
- LQLQRFGHAALLLE-UHFFFAOYSA-N 5-bromouracil Chemical compound BrC1=CNC(=O)NC1=O LQLQRFGHAALLLE-UHFFFAOYSA-N 0.000 description 1
- RHIULBJJKFDJPR-UHFFFAOYSA-N 5-ethyl-1h-pyrimidine-2,4-dione Chemical compound CCC1=CNC(=O)NC1=O RHIULBJJKFDJPR-UHFFFAOYSA-N 0.000 description 1
- KELXHQACBIUYSE-UHFFFAOYSA-N 5-methoxy-1h-pyrimidine-2,4-dione Chemical compound COC1=CNC(=O)NC1=O KELXHQACBIUYSE-UHFFFAOYSA-N 0.000 description 1
- FFKUHGONCHRHPE-UHFFFAOYSA-N 5-methyl-1h-pyrimidine-2,4-dione;7h-purin-6-amine Chemical compound CC1=CNC(=O)NC1=O.NC1=NC=NC2=C1NC=N2 FFKUHGONCHRHPE-UHFFFAOYSA-N 0.000 description 1
- ZLAQATDNGLKIEV-UHFFFAOYSA-N 5-methyl-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CC1=CNC(=S)NC1=O ZLAQATDNGLKIEV-UHFFFAOYSA-N 0.000 description 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- HDRIVHFYDVZHBY-UHFFFAOYSA-N 6-(methylaminomethyl)-4-oxo-2-sulfanylidene-1H-pyrimidine-5-carboxylic acid Chemical compound C(=O)(O)C=1C(NC(NC=1CNC)=S)=O HDRIVHFYDVZHBY-UHFFFAOYSA-N 0.000 description 1
- DCPSTSVLRXOYGS-UHFFFAOYSA-N 6-amino-1h-pyrimidine-2-thione Chemical compound NC1=CC=NC(S)=N1 DCPSTSVLRXOYGS-UHFFFAOYSA-N 0.000 description 1
- CKOMXBHMKXXTNW-UHFFFAOYSA-N 6-methyladenine Chemical compound CNC1=NC=NC2=C1N=CN2 CKOMXBHMKXXTNW-UHFFFAOYSA-N 0.000 description 1
- SHVCSCWHWMSGTE-UHFFFAOYSA-N 6-methyluracil Chemical compound CC1=CC(=O)NC(=O)N1 SHVCSCWHWMSGTE-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102100027211 Albumin Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-M Aminoacetate Chemical compound NCC([O-])=O DHMQDGOQFOQNFH-UHFFFAOYSA-M 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical group N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000288673 Chiroptera Species 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- BWLUMTFWVZZZND-UHFFFAOYSA-N Dibenzylamine Chemical class C=1C=CC=CC=1CNCC1=CC=CC=C1 BWLUMTFWVZZZND-UHFFFAOYSA-N 0.000 description 1
- XBPCUCUWBYBCDP-UHFFFAOYSA-N Dicyclohexylamine Chemical class C1CCCCC1NC1CCCCC1 XBPCUCUWBYBCDP-UHFFFAOYSA-N 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical class NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical class F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical class Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical class NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical class NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000000112 Myalgia Diseases 0.000 description 1
- SGSSKEDGVONRGC-UHFFFAOYSA-N N(2)-methylguanine Chemical compound O=C1NC(NC)=NC2=C1N=CN2 SGSSKEDGVONRGC-UHFFFAOYSA-N 0.000 description 1
- MBBZMMPHUWSWHV-BDVNFPICSA-N N-methylglucamine Chemical class CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO MBBZMMPHUWSWHV-BDVNFPICSA-N 0.000 description 1
- 241001292005 Nidovirales Species 0.000 description 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010010677 Phosphodiesterase I Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical class CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 1
- 101710198378 Uncharacterized 10.8 kDa protein in cox-rep intergenic region Proteins 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- SDFOCMGSQNWWPN-SXAUZNKPSA-N [2-[2-(diethylamino)ethylcarbamoyloxy]-3-[(z)-octadec-9-enoyl]oxypropyl] (z)-octadec-9-enoate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(OC(=O)NCCN(CC)CC)COC(=O)CCCCCCC\C=C/CCCCCCCC SDFOCMGSQNWWPN-SXAUZNKPSA-N 0.000 description 1
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 125000005600 alkyl phosphonate group Chemical group 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 125000000637 arginyl group Chemical class N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-L aspartate group Chemical class N[C@@H](CC(=O)[O-])C(=O)[O-] CKLJMWTZIZZHCS-REOHCLBHSA-L 0.000 description 1
- UPABQMWFWCMOFV-UHFFFAOYSA-N benethamine Chemical class C=1C=CC=CC=1CNCCC1=CC=CC=C1 UPABQMWFWCMOFV-UHFFFAOYSA-N 0.000 description 1
- JUHORIMYRDESRB-UHFFFAOYSA-N benzathine Chemical class C=1C=CC=CC=1CNCCNCC1=CC=CC=C1 JUHORIMYRDESRB-UHFFFAOYSA-N 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-M benzenesulfonate Chemical compound [O-]S(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-M 0.000 description 1
- 229940077388 benzenesulfonate Drugs 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 125000004063 butyryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- VDANGULDQQJODZ-UHFFFAOYSA-N chloroprocaine Chemical class CCN(CC)CCOC(=O)C1=CC=C(N)C=C1Cl VDANGULDQQJODZ-UHFFFAOYSA-N 0.000 description 1
- 150000001868 cobalt Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 150000001879 copper Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 125000001995 cyclobutyl group Chemical group [H]C1([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
- 125000000582 cycloheptyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 125000000640 cyclooctyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- 125000001511 cyclopentyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
- 125000001559 cyclopropyl group Chemical group [H]C1([H])C([H])([H])C1([H])* 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical class OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 150000005332 diethylamines Chemical class 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 208000017574 dry cough Diseases 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 150000002301 glucosamine derivatives Chemical class 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000002306 glutamic acid derivatives Chemical class 0.000 description 1
- 230000009422 growth inhibiting effect Effects 0.000 description 1
- 150000002357 guanidines Chemical class 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 125000003104 hexanoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000010842 high-capacity cDNA reverse transcription kit Methods 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000003978 infusion fluid Substances 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 159000000014 iron salts Chemical class 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 125000004491 isohexyl group Chemical group C(CCC(C)C)* 0.000 description 1
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229910003002 lithium salt Inorganic materials 0.000 description 1
- 159000000002 lithium salts Chemical class 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 229940049920 malate Drugs 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 150000002780 morpholines Chemical class 0.000 description 1
- 208000013465 muscle pain Diseases 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- XJVXMWNLQRTRGH-UHFFFAOYSA-N n-(3-methylbut-3-enyl)-2-methylsulfanyl-7h-purin-6-amine Chemical compound CSC1=NC(NCCC(C)=C)=C2NC=NC2=N1 XJVXMWNLQRTRGH-UHFFFAOYSA-N 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 1
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001038 naphthoyl group Chemical group C1(=CC=CC2=CC=CC=C12)C(=O)* 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 150000002815 nickel Chemical class 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 1
- 150000004885 piperazines Chemical class 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical class CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 229940043274 prophylactic drug Drugs 0.000 description 1
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000001973 tert-pentyl group Chemical group [H]C([H])([H])C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- QEMXHQIAXOOASZ-UHFFFAOYSA-N tetramethylammonium Chemical class C[N+](C)(C)C QEMXHQIAXOOASZ-UHFFFAOYSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000005425 toluyl group Chemical group 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical compound [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical class OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 125000003774 valeryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000006656 viral protein synthesis Effects 0.000 description 1
- 230000005727 virus proliferation Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Classifications
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- 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
Definitions
- the present invention provides antisense oligonucleotides having antiviral effects against SARS-CoV-2, SARS-CoV-1, or MERS-CoV, or pharmaceutically acceptable salts thereof, or hydrates thereof (hereinafter referred to as "the antisense oligonucleotide etc.”); pharmaceutical composition comprising said antisense oligonucleotide etc.; or treatment of viral infection and/or or related to preventive methods.
- COVID-19 (Coronavirus disease-2019) is a novel infectious disease characterized by pneumonia that led to the WHO declaring a pandemic in March 2020 after it was first identified in Wuhan, Hubei Province, China in November 2019.
- Non-Patent Document 1 The etiology of this COVID-19 is a novel virus, which was identified in January 2020 as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
- SARS-CoV-2 is evolutionarily closely related to SARS-CoV, the virus that caused the severe acute respiratory syndrome (SARS) epidemic in 2003. belongs to the genus (Non-Patent Document 2).
- SARS-CoV-2 The use of existing antiviral agents has been proposed for SARS-CoV-2, but at present, their effectiveness has not been clinically proven, and there is no established treatment. In addition, there are no established treatments for SARS-CoV-1 and MERS-CoV, which belong to the genus Betacoronavirus like SARS-CoV-2 and cause viral infections.
- the present invention provides an antisense oligonucleotide targeting a specific region of the SARS-CoV-2 genomic RNA described below, or a pharmaceutically acceptable salt or hydrate thereof, the antisense oligonucleotide, or A pharmaceutical composition or the like containing a pharmaceutically acceptable salt thereof or a hydrate thereof is provided.
- the antisense oligonucleotide of (1) or a pharmaceutically acceptable salt or their hydrates.
- At least one target region is the nsp1 region, nsp3 region, 3C-like proteinase region, nsp8 region, RNA-dependent RNA polymerase region, helicase region, 3'-to- selected from the group consisting of 5' exonuclease region, endoRNase region, 2'-O-ribose methyltransferase region, S region including S1 region and S2 region, M region, ORF7b region, N region, ORF10 region, and 3'UTR region
- the antisense oligonucleotide according to any one of (1) to (3), or a pharmaceutically acceptable salt or hydrate thereof.
- At least one target region is the nsp3 region, RNA-dependent RNA polymerase region, helicase region, 3'-to-5' exonuclease region, endoRNase region, 2'-O in the SARS-CoV-2 genomic RNA
- the antisense oligonucleotide according to any one of (1) to (4), which is selected from the group consisting of -ribose methyltransferase region, S region including S1 region and S2 region, and N region, or pharmaceutically acceptable thereof salts or their hydrates.
- Antisense oligonucleotide according to any one of the above, or a pharmaceutically acceptable salt or hydrate thereof.
- any one of (1) to (8) which is complementary to a nucleic acid comprising at least 10 consecutive bases of (10) Positions 6407-6426, 15173-15192, 15496-15515, 15498-15517, 16195-16214, 17084-17103, 20111-20130, 21502-21521 of SEQ ID NO: 1 , positions 28757 to 28776, and positions 28758 to 28777, which is complementary to a nucleic acid comprising at least 10 consecutive bases in at least one target region selected from the group consisting of any of (1) to (9). or a pharmaceutically acceptable salt or hydrate thereof.
- (12) (a) a base sequence selected from the group consisting of SEQ ID NOs: 2 to 177, (b) a nucleotide sequence in which one or several bases are added, deleted, or substituted in a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2-177, or (c) from the group consisting of SEQ ID NOS: 2-177
- the antisense oligonucleotide according to any one of (1) to (12) or a pharmaceutically acceptable salt or hydrate thereof, which inhibits the function of the target region.
- (14) (a) SEQ. a base sequence selected from the group consisting of 129, 131, 141, 157, 158, 162, 163, 164, and 165; (b) SEQ.
- 141, 157, 158, 162, 163, 164, and 165 in which one or several bases are added, deleted, or substituted in the base sequence selected from the group consisting of, or (c) SEQ ID NO: 9, 12, 36, 37, 61, 63, 64, 68, 70, 81, 82, 85, 88, 95, 96, 101, 106, 114, 119, 121, 124, 129, 131, 141, 157, comprising a nucleotide sequence having 80% or more sequence identity with a nucleotide sequence selected from the group consisting of 158, 162, 163, 164, and 165, and inhibiting the function of the target region, (1)- The antisense oligonucleotide according to any one of (13) or a pharmaceutically acceptable salt or hydrate thereof.
- (17-1) The antisense oligonucleotide according to any one of (1) to (16), which consists of 20 nucleotides, or a pharmaceutically acceptable salt or hydrate thereof.
- a gapmer in which the antisense oligonucleotide is composed of, from the 5′ side to the 3′ side, a wing region with a length of 5 nucleotides, a gap region with a length of 10 nucleotides, and a wing region with a length of 10 nucleotides is the antisense oligonucleotide according to (17-1) or a pharmaceutically acceptable salt or hydrate thereof.
- the wing region comprises a 2'-OMe (2'-O- CH3 ) group and/or a 2'-O-MOE ( 2' -O- CH2CH2OCH3 ) group, (1)-
- the bond between the second and third nucleosides and the bond between the fourth and fifth nucleosides from the 5′ side of the 5′ wing region are phosphodiester bonds, and from the 5′ side of the 3′ wing region
- the bond between the 1st and 2nd nucleosides and the bond between the 3rd and 4th nucleosides are phosphodiester bonds, and the bonds between other nucleosides are all phosphorothioate bonds, (17-1), (17 -2), or the antisense oligonucleotide according to (18), or a pharmaceutically acceptable salt or hydrate thereof.
- the bond between the second and third nucleosides, the bond between the third and fourth nucleosides, and the bond between the fourth and fifth nucleosides from the 5′ side of the 5′ wing region are phosphodiester bonds; and the bond between the first and second nucleosides, the bond between the second and third nucleosides, and the bond between the third and fourth nucleosides from the 5′ side of the 3′ wing region are phosphodiester bonds. and all other internucleoside bonds are phosphorothioate bonds, (17-1), (17-2), or the antisense oligonucleotide according to (18), or a pharmaceutically acceptable salt thereof, or them hydrate.
- a pharmaceutical composition comprising the antisense oligonucleotide according to any one of (1) to (20) or a pharmaceutically acceptable salt or hydrate thereof.
- the nucleic acid according to any one of (1) to (20) or a pharmaceutically acceptable salt or hydrate thereof, or the pharmaceutical composition according to any one of (21) to (22) A method for treating and/or preventing a viral infection selected from the group consisting of SARS-CoV-2, SARS-CoV-1, and MERS-CoV, comprising administering to a subject an effective amount of
- an antisense oligonucleotide that targets the genomic RNA of SARS-CoV-2 or a pharmaceutically acceptable salt or hydrate thereof, and the antisense oligonucleotide or a pharmaceutically acceptable A composition or the like containing a salt or a hydrate thereof is provided.
- the antisense oligonucleotide of the present invention can provide a therapeutic and/or prophylactic drug with high viral growth inhibitory effect and/or few side effects.
- the antisense oligonucleotides of the present invention are mutant strains of SARS-CoV-2 and/or known SARS-related coronaviruses such as SARS-CoV-1 (SARSr-CoV) and / Or it may also be effective against unknown SARS-related coronaviruses.
- the present invention provides antisense oligonucleotides or pharmaceutically acceptable salts or hydrates thereof having antiviral effects against SARS-CoV-2, SARS-CoV-1, or MERS-CoV (Hereinafter, the antisense oligonucleotide of the present invention, a pharmaceutically acceptable salt thereof, or a hydrate thereof will be collectively referred to as the "antisense oligonucleotide of the present invention").
- the antisense oligonucleotides of the invention consist of 15-30 nucleotides that are complementary to a nucleic acid containing at least 10 consecutive bases in the target region.
- the antisense oligonucleotides of the invention have at least 10, such as at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least Complementary to nucleic acids comprising or consisting of 20, such as 20 contiguous bases.
- an antisense oligonucleotide that is "complementary" to a nucleic acid is not limited to an antisense oligonucleotide that forms Watson-Crick base pairs with the nucleic acid of interest. Also includes antisense oligonucleotides that form a (wobble base pair).
- the Watson-Crick base pair means a base pair in which a hydrogen bond is formed between adenine - thymine, adenine - uracil, and guanine - cytosine. It refers to base pairs in which hydrogen bonds are formed between uracil, inosine-adenine, and inosine-cytosine.
- the "complementary base sequence” may not have 100% complementarity with the target base sequence.
- Non-complementary bases of bases, 4 bases, or 5 bases may be included, and base sequences that are 1 base, 2 bases, 3 bases, 4 bases, or 5 bases shorter than the target base sequence may be
- an antisense oligonucleotide that is "complementary" to a nucleic acid is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity.
- Complementarity can be readily determined by one skilled in the art, for example, by aligning two sequences, counting the number of bases that form Watson-Crick base pairs or wobble base pairs between the sequences, and determining the base pairs. It can be calculated by dividing the number of bases formed by the total number of bases in the sequence and multiplying this by 100.
- stringent conditions may be low stringent conditions, moderately stringent conditions, or highly stringent conditions.
- Low stringent conditions are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C.
- “moderately stringent conditions” are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 42°C or 5 x SSC, 1% SDS, 50 mM Tris-HCl (pH 7.0). 5), 50% formamide, 42°C.
- “Highly stringent conditions” are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. or 0.2 ⁇ SSC, 0.1% SDS, 65 ° C. be. Under these conditions, it can be expected that base sequences with higher sequence identity can be obtained more efficiently as the temperature is raised. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can appropriately select these factors. Similar stringency can be achieved by
- a commercially available kit for hybridization for example, AlkPhos Direct Labeling and Detection System (GE Healthcare) can be used.
- the membrane was washed with a primary washing buffer containing 0.1% (w/v) SDS at 55°C. After washing, hybridization can be detected.
- DIG digoxigenin
- a commercially available reagent e.g., PCR labeling mix (Roche Diagnostics)
- Hybridization can be detected using a DIG nucleic acid detection kit (Roche Diagnostics) or the like.
- the identity of the nucleotide sequence is determined using the algorithm BLAST (Basic Local Alignment Search Tool) by Carlin and Arthur (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993).
- BLAST Basic Local Alignment Search Tool
- Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990).
- BLAST and Gapped BLAST programs use the default parameters of each program.
- the antisense oligonucleotides of the present invention are, for example, 15 nucleotides or longer, 16 nucleotides or longer, 17 nucleotides or longer, 18 nucleotides or longer, 19 nucleotides or longer, 20 nucleotides or longer, 21 nucleotides or longer, 22 nucleotides or longer, 23 nucleotides or longer, 24 nucleotides or longer, 25 nucleotides or longer, 26 nucleotides or longer, 27 nucleotides or longer, 28 nucleotides or longer, 29 nucleotides or longer, or 30 nucleotides long.
- Antisense oligonucleotides of the invention may consist of, for example, 15-30 nucleotides, 15-25 nucleotides, 16-24 nucleotides, 17-23 nucleotides, 18-22 nucleotides, 19-21 nucleotides, eg 20 nucleotides.
- Examples of pharmaceutically acceptable salts of the antisense oligonucleotides of the invention include alkali metal salts such as sodium, potassium and lithium salts; alkaline earth metal salts such as calcium and magnesium salts; salts, metal salts such as iron salts, zinc salts, copper salts, nickel salts, cobalt salts; ammonium salts; t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, Organic amine salts such as tetramethylammoni
- sulfate perchlorate, sulfate, phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate; benzenesulfonate, p-toluene Aryl sulfonates such as sulfonates; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate; glycinate, lysine salts, amino acid salts such as arginine salts, ornithine salts, glutamates, aspartates, and the like. These salts can be produced by known methods. Alternatively, the antisense oligonucleotides of the invention may be in their hydrate form.
- the antisense oligonucleotides of the present invention are composed of nucleotides, and such nucleotides may be ribonucleotides, deoxyribonucleotides, or modified nucleotides.
- Modified nucleotides refer to those in which all or part of the nucleobases, sugar moieties, and phosphate-binding moieties that make up ribonucleotides or deoxyribonucleotides have been modified.
- Nucleic acid bases include, for example, adenine, guanine, hypoxanthine, cytosine, thymine, uracil, or modified bases thereof.
- modified bases include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (eg, 5-methylcytosine), 5-alkyluracil (eg, 5-ethyluracil), 5-halouracil (eg, , 5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (e.g., 6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxy methylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N
- thymine “T” and uracil “U” are interchangeable and either “T” or “U” essentially affects the activity of the antisense oligonucleotides of the invention. does not occur, the base sequences shown in this specification are indicated by the same SEQ ID NO, including cases where "T” is “U”.
- sequences containing modified bases are represented by the same SEQ ID numbers as sequences not containing modified bases. For example, “cytosine” and “methylcytosine” are interchangeable, and “cytosine” is " “Methylcytosine” is indicated by the same sequence number.
- Modifications of the sugar moiety can include, for example, modifications at the 2' position of ribose and modifications at other parts of the sugar.
- Modifications at the 2'-position of ribose include, for example, -OH group at the 2'-position of ribose with -OR, -OROR, -R, -R'OR, -SH, -SR, -NH 2 , -NHR, - NR 2 , —N 3 , —CN, —F, —Cl, —Br, —I, such as —OMe (—O—CH 3 ) or —Omethoxyethyl (—O—MOE: —O—CH 2 CH 2 OCH 3 ) can be mentioned.
- R represents alkyl, cycloalkyl, acyl or aryl.
- R' represents alkylene.
- Modifications of other parts of the sugar include, for example, those in which the 4'-position of ribose or deoxyribose is replaced with S, those in which the 2'-position and 4'-position of the sugar are crosslinked, for example, LNA (Locked Nucleic Acid ) or ENA (2′-O,4′-C-Ethylene-bridged Nucleic Acids), but are not limited thereto.
- LNA Locked Nucleic Acid
- ENA (2′-O,4′-C-Ethylene-bridged Nucleic Acids
- Modifications of the phosphate binding moiety include, for example, phosphodiester linkages, phosphorothioate linkages, phosphorodithioate linkages, alkylphosphonate linkages, phosphoramidate linkages, boranophosphate linkages (e.g., Enya et al: Bioorganic & Medicinal Chemistry , 2008, 18, 9154-9160) (see, eg, Republished Patent Publication No. 2006/129594 and Republished Patent Publication No. 2006/038608).
- alkyl is preferably linear or branched alkyl having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl and isohexyl. be done.
- the alkyl may be substituted, and examples of such substituents include halogen, alkoxy, cyano, and nitro, and may be substituted with 1 to 3 of these.
- cycloalkyl having 3 to 12 carbon atoms is preferred as cycloalkyl.
- Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl.
- halogen includes fluorine, chlorine, bromine, and iodine.
- alkoxy refers to linear or branched alkoxy having 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert. -butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, isohexyloxy and the like.
- Alkoxy having 1 to 3 carbon atoms is particularly preferred.
- aryl having 6 to 10 carbon atoms is preferred as aryl.
- Specific examples include phenyl, ⁇ -naphthyl, and ⁇ -naphthyl. Phenyl is particularly preferred.
- the aryl may be substituted, and examples of such substituents include alkyl, halogen, alkoxy, cyano, and nitro, which may be substituted 1-3 times.
- alkylene is preferably linear or branched alkylene having 1 to 6 carbon atoms.
- Specific examples include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, 2-(ethyl)trimethylene, and 1-(methyl)tetramethylene.
- acyl includes linear or branched alkanoyl or aroyl.
- Alkanoyl includes, for example, formyl, acetyl, 2-methylacetyl, 2,2-dimethylacetyl, propionyl, butyryl, isobutyryl, pentanoyl, 2,2-dimethylpropionyl, hexanoyl and the like.
- Aroyl includes, for example, benzoyl, toluoyl and naphthoyl. Such aroyl may be optionally substituted at any substitutable position or may be substituted with alkyl.
- the present invention provides that the antisense oligonucleotide comprises a central gap region and two wing regions flanking the 5′ and 3′ ends of the gap region (5′ wing region and 3′ wing region, respectively). are also described as gapmers.
- a gap region is a region recognized by RNase H and is composed of deoxyribonucleotides with unmodified sugar moieties.
- the wing region contains at least one modified nucleotide, for example, all of which are composed of modified nucleotides (eg, ribonucleotides modified at the 2' position of ribose).
- the nucleosides of the 5' and 3' wing regions each have at least one, such as two or more, three or more, four or more, five or more sugar moiety modifications, such as a 2'-OMe group. and/or contain a 2'-O-MOE group, for example all of the nucleosides in the 5' and 3' wing regions may contain a 2'-OMe group and/or a 2'-O-MOE group. .
- the nucleosides of the 5' wing region and 3' wing region may contain modifications in the base portion, such as containing at least one methylcytosine.
- the length of the gap region is not limited, but may be, for example, 5-15, 8-12, 9-11 or 10 bases long.
- the length of the 5' wing region and the 3' wing region is not limited, but may be, for example, each independently 2-10, 3-8, 4-6 or 5 bases long.
- the structure of a gapmer is sometimes referred to herein as "a-b-c type".
- "a-b-c type" means that a gapmer is composed of a wing region of nucleotide length a, a gap region of nucleotide length b, and a wing region of nucleotide length c from the 5' side to the 3' side. do.
- gapmers are gaps of type 5-10-5, 4-10-5, 5-10-4, 4-10-4, 5-9-4, or 4-9-4. May be Ma.
- gapmers of the invention comprise modifications of one or more phosphate binding moieties, such as phosphorothioate linkages, such as one or more, two or more, three or more, four or more, five internucleotide linkages. One or more, ten or more, fifteen or more, such as all may be phosphorothioate linkages.
- the gapmers of the invention have 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more , or ten or more (eg, 4-6), eg, in the 5′ wing region and/or the 3′ wing region, and all other internucleoside linkages are phosphorothioate linkages.
- the linkage between the 2nd and 3rd nucleosides and the linkage between the 4th and 5th nucleosides from the 5' side of the 5' wing region are phosphodiester linkages, and the 5' The bond between the first and second nucleosides and the bond between the third and fourth nucleosides from the side are phosphodiester bonds, and the bonds between other nucleosides are all phosphorothioate bonds.
- the antisense oligonucleotides of the invention are directed against SARS-CoV-2, SARS-CoV-1 or MERS-CoV, such as SARS-CoV-2 or SARS-CoV-1, such as SARS-CoV-2 Has antiviral effect.
- SARS-CoV-2 includes a virus having a genomic RNA sequence consisting of the nucleotide sequence NC — 045512.2 (SEQ ID NO: 1) or a mutant strain thereof.
- SARS-CoV-1 includes viruses having a genomic RNA sequence consisting of the nucleotide sequence of NC_004718.3 or variants thereof.
- MERS-CoV includes a virus having a genomic RNA sequence consisting of the base sequence of NC_019843.3 or a mutant strain thereof.
- mutant strains refer to offspring with new properties that are created by mutations that partially change genetic information due to partial misreading and recombination during the replication process of viral genes. Mutants have altered some properties affected by altered genetic information, but the original virus species remains unchanged.
- coronavirus family if 90% or more of the amino acid sequence of the conserved replicase region is shared, it is said to belong to the same virus species (ICTV 9th report (2011) nidovirales ), Coronaviridae (see https://talk.ictvonline.org/ictv-reports/ictv_9th_report/positive-sense-rna-viruses-2011/w/posrna_viruses/222/coronaviridae)).
- the antisense oligonucleotides of the present invention can be effective against not only known SARS-related coronaviruses (SARSr-CoV) but also unknown SARS-related coronaviruses.
- SARS-related coronavirus is a type of coronavirus that infects mammals, including humans and bats, and is an enveloped virus belonging to the genus betacoronavirus (group 2 coronavirus). It refers to a main-strand positive-strand RNA virus.
- group 2 coronavirus refers to a main-strand positive-strand RNA virus.
- the SARS-associated coronavirus utilizes the angiotensin-converting enzyme 2 (ACE2) receptor to enter cells.
- ACE2 angiotensin-converting enzyme 2
- the antiviral effect means an effect of suppressing viral proliferation and/or reducing viral infectivity. Whether or not the antisense oligonucleotides of the present invention have antiviral effects can be tested as described in the Examples of the present specification. For example, the presence or absence of an antiviral effect can be determined by preparing a plasmid that expresses a nucleic acid containing a target sequence, introducing the plasmid and the antisense oligonucleotide of the present invention into a cell, and obtaining the amount of the nucleic acid containing the target sequence expressed from the cell.
- the presence or absence of the antiviral effect can be determined by introducing the virus and the antisense oligonucleotide of the present invention into cells and culturing them, and then determining whether the amount of the virus in the cell or the cell culture supernatant or the nucleic acid derived therefrom is higher than that of the negative control nucleic acid. It can be measured by examining whether it decreases (for example, 5% or more, 10% or more, or 20% or more) compared to when it is introduced.
- the antisense oligonucleotides of the invention are tested for gapmer knockdown activity using a SARS-CoV-2 expression plasmid at either 1 ⁇ M or 3 ⁇ M when tested as described in Example 1. 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90 % knockdown activity or have a target sequence identical to an antisense oligonucleotide that has these knockdown activities.
- the antisense oligonucleotides of the invention show 2, 3, 6, 8 , or at any concentration of 10 ⁇ M, ⁇ 5%, ⁇ 10%, ⁇ 20%, ⁇ 30%, ⁇ 40%, ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90% It has knockdown activity or has the same target sequence as an antisense oligonucleotide that has these knockdown activities.
- the antisense oligonucleotides of the present invention are the 5'UTR region, nsp1 region, nsp3 region, nsp4 region, 3C-like proteinase region, nsp6 region, nsp7 region, nsp8 region, and nsp9 region in the SARS-CoV-2 genomic RNA.
- nsp10 region RNA-dependent RNA polymerase region, helicase region, 3'-to-5' exonuclease region, endoRNase region, 2'-O-ribose methyltransferase region, S region including S1 region and S2 region, ORF3a region, E A sequence in at least one target region selected from the group consisting of region, M region, ORF7b region, N region, ORF10 region, and 3'UTR region is targeted.
- sequence of the SARS-CoV-2 genomic RNA and the sequence of each region in the genomic RNA can be easily determined by referring to databases (eg, ncbi).
- the sequence of the genomic RNA of SARS-CoV-2 may be the base sequence of NC_045512.2 (SEQ ID NO: 1).
- the 5'UTR region is the nucleotide sequence of positions 1 to 265 of SEQ ID NO: 1
- the nsp1 region is the nucleotide sequence of positions 266 to 805 of SEQ ID NO: 1
- the nsp3 region is the sequence of SEQ ID NO: 1.
- nsp4 region is 8555-10054 base sequence of SEQ ID NO: 1
- 3C-like proteinase region is 10055-10972 base sequence of SEQ ID NO: 1
- nsp6 region is SEQ ID NO The nucleotide sequence of positions 10973 to 11842 of 1
- the nsp7 region is the nucleotide sequence of positions 11843 to 12091 of SEQ ID NO: 1
- the nsp8 region is the nucleotide sequence of positions 12092 to 12685 of SEQ ID NO: 1
- the nsp9 region is of SEQ ID NO: 1
- Nucleotide sequence from positions 12686 to 13024, nsp10 region from 13025 to 13441 of SEQ ID NO: 1, RNA-dependent RNA polymerase region from 13442 to 16236 of SEQ ID NO: 1, helicase region from SEQ ID NO: 1, the 3'-to-5' exonuclease region is the nucleotide
- the antisense oligonucleotides of the invention target regions that are conserved in the sequences of SARS-CoV-2 and SARS-CoV-1 genomic RNAs, and thus SARS-CoV- 2. It can have antiviral effects against betacoronaviruses, including SARS-CoV-1 and MERS-CoV.
- the antisense oligonucleotide of the present invention is a region conserved in the sequences of the genomic RNA of SARS-CoV-2 and the genomic RNA of SARS-CoV-1, among the above target regions.
- the antisense oligonucleotides of the invention are at positions 47-66, 242-261, 259-278, 292-311, 456-475, 704 of SEQ ID NO:1. ⁇ 723rd, 3353rd ⁇ 3372nd, 5384th ⁇ 5403rd, 6071st ⁇ 6090th, 6406th ⁇ 6425th, 6407th ⁇ 6426th, 6408th ⁇ 6427th, 6797th ⁇ 6815th, 7532nd ⁇ 7551th 8707th to 8724th, 10292nd to 10309th, 10407th to 10426th, 10409th to 10428th, 10487th to 10506th, 11609th to 11628th, 12025th to 12044th, 12170th to 12188th, 12214-12233, 12316-12335, 12401-12420, 12589-12608, 12843-12862, 12900-12919, 12969-12988, 13
- the antisense oligonucleotides of the invention are (a) a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2-177; (b) a nucleotide sequence in which one or several bases are added, deleted, or substituted in a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2-177, or (c) from the group consisting of SEQ ID NOS: 2-177 80%, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% of the selected nucleotide sequence 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of sequence identity, or consists of any of these sequences.
- the antisense oligonucleotide of the present invention comprises or consists of any one of the base sequences of (a) above.
- the target region of at least one of the antisense oligonucleotides of the present invention is the nsp1 region, nsp3 region, 3C-like proteinase region, nsp8 region, RNA-dependent RNA polymerase region in SARS-CoV-2 genomic RNA. region, helicase region, 3'-to-5' exonuclease region, endoRNase region, 2'-O-ribose methyltransferase region, S region including S1 region and S2 region, M region, ORF7b region, N region, ORF10 region, and Selected from the group consisting of the 3'UTR region.
- the antisense oligonucleotides of the invention are located at positions 704-723, 5384-5403, 6407-6426, 10409-10428, 10487-10506, 12170-12188 of SEQ ID NO:1.
- the antisense oligonucleotides of the invention are (a) SEQ. , 85, 88-90, 92, 93, 95, 96, 99, 101, 106-110, 112-114, 117, 119, 121, 124, 125-127, 129, 131, 138-143, 149, 152 , 153, 155, 157, 158, 162-165, 167-169, and 171-176. (b) SEQ.
- the antisense oligonucleotide of the present invention comprises or consists of any one of the base sequences of (a) above.
- the target region of at least one of the antisense oligonucleotides of the present invention is the nsp3 region, RNA-dependent RNA polymerase region, helicase region, 3'-to-5' region in the genomic RNA of SARS-CoV-2. It is selected from the group consisting of exonuclease region, endoRNase region, 2'-O-ribose methyltransferase region, S region including S1 region and S2 region, and N region.
- the antisense oligonucleotides of the invention are located at positions 5384-5403, 6407-6426, 13642-13661, 13672-13691, 15173-15192, 15175-15194 of SEQ ID NO:1.
- the antisense oligonucleotides of the invention are located at positions 6407-6426, 15173-15192, 15496-15515, 15498-15517, 16195-16214, 17084-17103 of SEQ ID NO
- the antisense oligonucleotides of the invention are (a) SEQ. , 141, 157, 158, 162, 163, 164, and 165. (b) SEQ. , 141, 157, 158, 162, 163, 164, and 165 in which one or several bases are added, deleted, or substituted in the base sequence selected from the group consisting of, or (c) SEQ ID NO: 9, 12, 36, 37, 61, 63, 64, 68, 70, 81, 82, 85, 88, 95, 96, 101, 106, 114, 119, 121, 124, 129, 131, 141, 157, A base sequence having 80% or more sequence identity to a base sequence selected from the group consisting of 158, 162, 163, 164, and 165, or consists of any one of these sequences.
- the antisense oligonucleotide of the present invention comprises or consists of any one of the base sequences of (a) above.
- the antisense oligonucleotides of the invention are (a) a nucleotide sequence selected from the group consisting of SEQ ID NOS: 12, 61, 68, 70, 85, 96, 124, 129, 163, and 164; (b) one or several bases are added, deleted, or substituted in the base sequence selected from the group consisting of SEQ ID NOs: 12, 61, 68, 70, 85, 96, 124, 129, 163, and 164 or (c) 80% or more, 85% or more of the base sequence selected from the group consisting of SEQ ID NOs: 12, 61, 68, 70, 85, 96, 124, 129, 163, and 164 , 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more
- the antisense oligonucleotides of the invention inhibit the function of the target region.
- inhibiting the function of the target region means that RNA containing the target region bound to the target region and formed a double strand with the antisense oligonucleotide is cleaved by RNaseH, and the target region is This includes one or more of inhibiting replication of the RNA containing the target region, inhibiting translation of the target region if it is translated, and inhibiting transcription of the RNA containing the target region.
- the target region may exist in subgenomic RNA as well as in genomic RNA, and "subgenomic RNA” is synthesized by RNA-dependent RNA polymerase using (+) strand genomic RNA as a template. It is an RNA that is shorter than the genomic RNA synthesized by RNA-dependent RNA polymerase using a part of the (-) strand of RNA as a template, and serves as mRNA for viral protein synthesis (translation).
- the antisense oligonucleotides of the present invention are generally described in W.W. Brad Wan et al., Nucleic Acid Research, Vol. 42, No. 22 13456 (2014) and the like. In addition, it can be easily synthesized using various automatic synthesizers (e.g., AKTA oligopilot plus 10/100 (GE Healthcare)), or by third-party organizations (e.g., Promega or Takara). It can also be manufactured on commission.
- various automatic synthesizers e.g., AKTA oligopilot plus 10/100 (GE Healthcare)
- third-party organizations e.g., Promega or Takara
- the present invention relates to pharmaceutical compositions comprising one or more of the antisense oligonucleotides of the present invention or pharmaceutically acceptable salts or hydrates thereof.
- the pharmaceutical compositions of the invention may contain carriers that facilitate delivery of the antisense nucleotides.
- Such carriers are not particularly limited as long as they are pharmaceutically acceptable, and examples thereof include cationic carriers such as cationic liposomes and cationic polymers, and carriers utilizing virus envelopes. .
- Cationic liposomes include, for example, liposomes formed from 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoylglycerol and phospholipids as essential constituents (hereinafter, “liposome A” ), Oligofectamine (registered trademark) (manufactured by Invitrogen), Lipofectin (registered trademark) (manufactured by Invitrogen), Lipofectamine (registered trademark) (manufactured by Invitrogen), Lipofectamine 2000 (registered trademark) (manufactured by Invitrogen ), DMRIE-C (registered trademark) (manufactured by Invitrogen), GeneSilencer (registered trademark) (manufactured by Gene Therapy Systems), TransMessenger (registered trademark) (manufactured by QIAGEN), TransIT TKO (registered trademark) (manufactured by Mir
- cationic polymers examples include JetSI (registered trademark) (manufactured by Qbiogene) and Jet-PEI (registered trademark) (polyethyleneimine, manufactured by Qbiogene).
- Carriers using viral envelopes include, for example, GenomeOne (registered trademark) (HVJ-E liposome, manufactured by Ishihara Sangyo Co., Ltd.).
- GenomeOne registered trademark
- HVJ-E liposome manufactured by Ishihara Sangyo Co., Ltd.
- the antisense oligonucleotides of the present invention may be conjugated with lipids and the like in pharmaceutical compositions in order to facilitate delivery of the antisense oligonucleotides.
- it can be conjugated with cholesterol as described in Bijsterbosch, M.K. et al. (2000) Nucleic Acid Res., 28, 2717-2725.
- the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable additive in addition to the antisense oligonucleotide or a pharmaceutically acceptable salt or hydrate thereof and optionally the above carrier.
- a pharmaceutically acceptable additive include, for example, emulsifying aids (e.g. C6-22 fatty acids and pharmaceutically acceptable salts thereof, albumin, dextran), stabilizers (e.g. cholesterol, phosphatidic acid, mannitol, sorbitol). , tonicity agents (e.g. sodium chloride, glucose, maltose, lactose, sucrose, trehalose), pH adjusters (e.g.
- hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, triethanolamine can be mentioned. 1 type, or 2 or more types can be used for these.
- the content of the additive in the composition of the present invention is suitably 90% by weight or less, preferably 70% by weight or less, more preferably 50% by weight or less.
- the method for preparing the pharmaceutical composition of the present invention is not limited, it can be prepared, for example, by adding the antisense oligonucleotide of the present invention to a carrier dispersion and stirring appropriately. Moreover, the additive can be added in an appropriate step before or after the addition of the antisense oligonucleotide of the present invention.
- the aqueous solvent that can be used when adding the antisense oligonucleotide of the present invention is not particularly limited as long as it is pharmaceutically acceptable. For example, water for injection, distilled water for injection, electrolytes such as physiological saline liquid, glucose liquid, sugar liquid such as maltose liquid. Moreover, conditions such as pH and temperature in such a case can be appropriately selected by those skilled in the art.
- the pharmaceutical composition of the present invention can be, for example, a liquid formulation or a freeze-dried formulation thereof.
- the freeze-dried preparation can be prepared by freeze-drying the composition of the present invention in the form of a liquid formulation by a conventional method. For example, after appropriately sterilizing the composition of the present invention in the form of a liquid formulation, a predetermined amount is dispensed into vials and pre-frozen under conditions within the range of about -40°C to -20°C. for about 2 hours, primary drying under reduced pressure within the range of about 0 ° C. to 10 ° C., then secondary drying under reduced pressure within the range of about 15 ° C. to 25 ° C. It can be freeze-dried. . Then, in general, the inside of the vial is replaced with nitrogen gas and capped to obtain a freeze-dried preparation of the composition of the present invention.
- the freeze-dried formulation of the pharmaceutical composition of the present invention can generally be redissolved by adding any suitable solution (redissolving solution) before use.
- suitable solution redissolving solution
- suitable solution include water for injection, physiological saline, and other general infusion solutions.
- the volume of this re-dissolution solution is not particularly limited depending on the application and the like, but it is suitable to be 0.5 to 2 times the volume of the liquid before freeze-drying or 500 mL or less.
- the dosage for administering the composition according to the present invention includes the type of antisense oligonucleotide according to the present invention to be contained or a pharmaceutically acceptable salt thereof or a hydrate thereof, dosage form of the composition,
- the amount of the antisense oligonucleotide of the present invention is, for example, 0.01 mg per 1 kg of body weight per administration, although it can be adjusted in consideration of the patient's condition such as age and weight, route of administration, nature and symptoms of the disease. ⁇ 200 mg or 0.1 mg to 20 mg, preferably 0.2 mg to 10 mg/kg body weight, more preferably 0.5 mg to 4 mg/kg body weight, more preferably 1 mg to 2 mg/kg body weight.
- the number and frequency of administration are not limited, but for example, it can be administered only once, and several days later (for example, the next day to within 1 week), it can be administered one or more times for a total of multiple times (for example, a total of 2 times). ) can also be administered.
- the dosing frequency may be once every 1-3 days, once every 4-6 days, once every week, or once every 2-3 weeks.
- the numerical value here may vary depending on the type of target disease, dosage form, and target molecule. Accordingly, a lower dose or administration frequency may be sufficient in some cases, and conversely, a higher dose or administration frequency may be required.
- the dosage form of the composition according to the present invention is not particularly limited as long as it is a pharmaceutically acceptable dosage form, and can be selected according to the treatment method. , intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, oral administration, intratissue administration, transdermal administration and the like. Moreover, the dosage form that the composition of the present invention can take is not particularly limited, but examples thereof include inhalants, various injections, oral agents, infusions, ointments, lotions, and the like.
- the dosage form of the pharmaceutical composition according to the present invention is preferably intratracheal administration, and the dosage form that the composition of the present invention can take is preferably an inhalant.
- nebulizer powder inhalants (eg DPI (dry powder inhaler) administration), aerosols, preferably inhalable liquids.
- Subjects to which the antisense oligonucleotides or pharmaceutical compositions of the present invention are administered are, for example, mammals such as primates such as humans, laboratory animals such as rats, mice, and rats, and livestock animals such as pigs, cows, horses, and sheep. etc., preferably human.
- the present invention provides an antisense oligonucleotide of the invention, or a pharmaceutically acceptable salt or hydrate thereof, or a pharmaceutical composition, comprising administering to a subject a SARS-CoV- 2.
- the pharmaceutical composition, dosage amount, administration route, etc. of the pharmaceutical composition in this embodiment are as described herein.
- the treatment of viral infections refers to alleviation, amelioration, and remission of diseases caused by the above viruses or symptoms thereof (e.g., one or more of dyspnea, fever, dry cough, headache, chills, and muscle pain). includes one or more of As used herein, prevention of viral infections includes reduction of the risk of developing diseases or symptoms thereof caused by the above viruses.
- Example 1 Measurement of gapmer knockdown activity using SARS-CoV-2 expression plasmid>
- the sequences of SARS-CoV-2 (reference sequence; NC_045512.2) (SEQ ID NO: 1) and SARS-CoV-1 (reference sequence; NC_004718.3) genomic RNA sequences were compared and conserved between the two species. identified areas where Regions conserved between the two species include positions 43 to 89, positions 242 to 279, positions 290 to 312, positions 455 to 477, positions 704 to 723, positions 3352 to 3372, and positions 5384 to 5403 of SEQ ID NO: 1.
- test substances gapmer Nos. G1 to G176 (SEQ ID NOs: 2 to 177) were prepared. All nucleosides in the wing regions of test substance gapmer Nos. G1 to G176 contain 2'-OMe (2'-O-CH 3 ) groups.
- the gapmer with "MOE" at the end of the gamper No. is a 2'-O-MOE gapmer (all nucleosides in the wing portion are 2'-O-MOE (2'-O-CH 2 CH 2 OCH 3 ) containing gapmers).
- all bonds between nucleosides are phosphorothioate bonds.
- all oligonucleotides are, in principle, 5-10-5 type gapmers, except for the following oligonucleotides.
- 19mer 4-10-5 type G110, G114 19mer 5-10-4 type: G11, G19, G61, G115 18mer 4-10-4 type: G13, G14, G81, G84, G85, G88, G90 18mer 5-9-4 type: G117 17mer 4-9-4 type: 133-2'OMe, 133-2'MOE, 134-2'OMe, 134-2'MOE
- the "abc type” means that the gapmer consists of a wing region of nucleotide length a, a gap region of nucleotide length b, and a wing region of nucleotide length c from the 5' side to the 3' side.
- two target sequences (5′UTR/nsp1 of G3) means that the Gapmer target sequence contains two regions.
- the bond between the 2nd and 3rd nucleosides and the bond between the 4th and 5th nucleosides from the 5' side of the 5' wing region are phosphodiester bonds.
- the bond between the nucleosides is a phosphodiester bond, and the bond between the 1st and 2nd nucleosides, the 2nd and 3rd nucleosides, and the 3rd and 4th from the 5' side of the 3′ wing region.
- the first internucleoside bond is a phosphodiester bond, and all other internucleoside bonds are phosphorothioate bonds (PO/PS type-1).
- SARS-CoV-2 expression plasmid at a final concentration of 400 ng/sample, Firefly luciferase (Fluc) expression plasmid (control plasmid) at a final concentration of 4 ng/sample, test substance gapmer at a final concentration of 1, 3 ⁇ M or negative control gapmer, 293T cells (obtained from ATCC), using SF Cell Line 4D-Nucleofector X Kit (Lonza) and 4D-Nucleofector (Lonza) (Nuleofector 16.4 ⁇ L and supplement 3.6 ⁇ L), DS from the pulse program installed in 4D-Nucleofector -130 was selected and electroporated.
- the SARS-CoV-2 expression plasmid was prepared by dividing the SARS-CoV-2 genomic RNA into 9 parts (Table 1-3), synthesizing it as a DNA artificial gene, and subcloning it into the expression plasmid pcDNA3.1. Artificial gene synthesis and subcloning into expression plasmids were outsourced to Thermo Fisher Scientific. However, since plasmid No.
- SARS-CoV-2 (2019-nCoV) 3C-like proteinase/3CLpro Gene ORF cDNA clone expression plasmid (Sino Biological) and SARS -CoV-2 (2019-nCoV) nsp8 Gene ORF cDNA clone expression plasmid (Sino Biological) was used (respectively described as "3CLpro” and "nsp8" in Tables 1-3).
- As a control plasmid pGL4.50 [luc2/CMV/Hygro] Vector (Promega) was used. The sequence of ASO#129700 (Wheeler et al. 2012. Nature 488: 111-5.
- test substance gapmer and the negative control gapmer are 2'-OMe gapmer (a gapmer in which all nucleosides of the wing portion contain 2'-OMe (2'-O- CH3 )) or a 2'-O-MOE gapmer (a wing portion All of the nucleosides of 2'-O-MOE (2'-O- CH2CH2OCH3 ) were synthesized as gapmers containing 2' -O- CH2CH2OCH3 .
- a reverse transcription (RT) reaction was performed using Using Fast SYBR Green Master Mix (Thermo Fisher Scientific), qPCR was performed using the RT reaction mixture as a template according to the manufacturer's protocol, and the SARS-CoV-2 RNA expression level derived from the SARS-CoV-2 expression plasmid was measured.
- Primers used for detection of SARS-CoV-2 RNA from SARS-CoV-2 expression plasmids are shown in Table 1-3.
- the SARS-CoV-2 RNA expression level was corrected with the Fluc RNA expression level derived from the control plasmid or the PPIB RNA expression level of the housekeeping gene. Primers used for detection of Fluc RNA or PPIB RNA are shown in Table 1-4.
- the SARS-CoV-2 RNA expression level suppression rate (knockdown activity of the test substance gapmer) when the test substance gapmer was introduced relative to the SARS-CoV-2 RNA expression level when the negative control gapmer was introduced was analyzed. Results are shown in Table 1-5.
- Fluc-corrected 5' side and “Fluc-corrected 3' side” refer to the 5' side of the two pairs of detection primers (Table 1-3), 5' side and 3' side. or the 3' side was used to detect expression.
- Example 2 Measurement of gapmer knockdown activity using SARS-CoV-2 virus> Test substance gapmer or negative control gapmer at a final concentration of 2-10 ⁇ M was added to human ACE2 stably expressing cells (293T-ACE2) prepared by infecting 293T cells with a human ACE2-expressing lentiviral vector and injected with Amxa Cell Line Nucleofector Kit V (Lonza ) (solution V 73.6 uL and supplement 16.4 uL/sample) and NucleofectorII (Amaxa) were used, A-023 was selected from the pulse program installed in NucleofectorII, and electroporation was performed. The sequence of ASO#129700 (Wheeler et al. 2012.
- test substance gapmer is the sequence of Gapmer Nos. 1 to 135 used in Example 1, which was partially unevaluated, and Gapmer No. 136, which was additionally designed based on the activity data of Gapmer No. 1 to 135. G176 was used.
- the test substance gapmer and the negative control gapmer were synthesized as 2'-OMe gapmer or 2'-MOE gapmer by Nippon Bio Service or Ajinomoto Bio-Pharma Service. 293T-ACE2 used 6.0 ⁇ 10 5 cells per electroporation.
- the SARS-CoV-2 virus is based on the WK521 strain obtained from the National Institute of Infectious Diseases and described in the literature (Terada et al. 2019. J Virol 93: e01208-19. doi: 10.1128/JVI.01208-19).
- a recombinant SARS-CoV-2 virus (rSARS-CoV-2-ORF8-Nluc) with a Nano-luciferase gene integrated into the coding region of the accessory protein ORF8 was used using a similar method.
- One hour after adding the virus solution the virus in the medium was removed by exchanging the medium.
- After 48 hours from virus infection the culture supernatant was collected, and a cell lysate was prepared using Passive Lysis Buffer (Promega).
- the Luciferase Assay System Promega
- the luciferase activity in the cell lysate was measured according to the attached protocol, and the intracellular SARS-CoV-2 viral load was evaluated (measurement of intracellular viral load).
- the recovered culture supernatant was added to 293T-ACE2 cells separately seeded at 3.0 ⁇ 10 4 cells/well in a 96-well plate at 5 ⁇ L/well to infect the cells with the virus in the culture supernatant.
- the intracellular SARS-CoV-2 viral load was evaluated in the same manner as for the first virus infection, and the infectious virus load in the culture supernatant was evaluated (infectious virus in medium volume measurement).
- Table 2 shows the knockdown activity of the test substance gapmer (infectious virus amount suppression rate in the medium: the infectious virus amount suppression rate when the test substance gapmer was introduced relative to the infectious virus amount when the negative control gapmer was introduced).
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Public Health (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Virology (AREA)
- Communicable Diseases (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
SARS-CoV-2、SARS-CoV-1、及びMERS-CoVからなる群から選択されるウイルスに対する抗ウイルス効果を有する、アンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(2)アンチセンスオリゴヌクレオチドが中央のギャップ領域、及びギャップ領域に隣接する2つのウイング領域を含むギャップマーである、(1)に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(3)ウイルスがSARS-CoV-2又はSARS-CoV-1である、(1)又は(2)に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(4)少なくとも一つの標的領域が、SARS-CoV-2のゲノムRNAにおける、nsp1領域、nsp3領域、3C-like proteinase領域、nsp8領域、RNA-dependent RNA polymerase領域、helicase領域、3'-to-5' exonuclease領域、endoRNase領域、2'-O-ribose methyltransferase領域、S1領域及びS2領域を含むS領域、M領域、ORF7b領域、N領域、ORF10領域、及び3'UTR領域からなる群から選択される、(1)~(3)のいずれかに記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(5)少なくとも一つの標的領域が、SARS-CoV-2のゲノムRNAにおける、nsp3領域、RNA-dependent RNA polymerase領域、helicase領域、3'-to-5' exonuclease領域、endoRNase領域、2'-O-ribose methyltransferase領域、S1領域及びS2領域を含むS領域、及びN領域からなる群から選択される、(1)~(4)のいずれかに記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(6)配列番号1の43位~89位、242~279位、290~312位、455~477位、704~723位、3352位~3372位、5384~5403位、6071~6090位、6406位~6427位、6797~6815位、7532~7551位、8707~8724位、10292~10309位、10406位~10431位、10484位~10506位、11609~11630位、12023~12045位、12170~12188位、12212~12234位、12314~12339位、12401~12420位、12589~12608位、12839位~12867位、12898位~12922位、12965位~12990位、13151~13175位、13271~13290位、13363~13386位、13458位~13502位、13642~13661位、13672~13691位、13762位~13790位、13894位~13916位、14050~14069位、14290位~14312位、14512~14531位、14654位~14687位、14698~14717位、14750位~14777位、14824位~14846位、14854~14873位、14878位~14909位、14953位~14990位、14992位~15026位、15037位~15061位、15063位~15140位、15172位~15198位、15278位~15299位、15454位~15479位、15496位~15518位、15520~15539位、15622位~15644位、15791~15809位、15829位~15859位、15886~15905位、15929位~15950位、15985~16011位、16051位~16085位、16189位~16220位、16430位~16451位、16636~16655位、16822位~16845位、17015位~17051位、17080位~17103位、17137~17156位、17215~17234位、17254位~17277位、17564位~17600位、17748~17765位、17773~17792位、17830~17849位、17859~17876位、18094~18111位、18196位~18218位、18253位~18278位、18370~18387位、19568位~19595位、19622~19639位、19780~19802位、20107~20130位、20776~20795位、20797~20816位、20888位~20909位、21453~21472位、21502位~21524位、22550~22569位、22814位~22836位、23093位~23113位、23956位~23976位、24302位~24324位、24446~24465位、24467位~24489位、24620位~24651位、24662位~24684位、24962位~24982位、25104位~25128位、25364位~25384位、25502~25520位、26287位~26325位、26574位~26604位、26782~26800位、27093~27111位、27771~27794位、27806~27823位、28270位~28294位、28397位~28418位、28509位~28538位、28744位~28784位、28799位~28820位、28946位~28972位、28986~29005位、29010位~29031位、29102位~29130位、29174位~29196位、29354~29373位、29615~29634位、29712~29731位、及び29787~29867位からなる群から選択される少なくとも一つの標的領域中の少なくとも10の連続する塩基を含む核酸に相補的である、(1)~(5)のいずれかに記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(7)配列番号1の47位~66位、242位~261位、259位~278位、292位~311位、456位~475位、704位~723位、3353位~3372位、5384位~5403位、6071位~6090位、6406位~6425位、6407位~6426位、6408位~6427位、6797位~6815位、7532位~7551位、8707位~8724位、10292位~10309位、10407位~10426位、10409位~10428位、10487位~10506位、11609位~11628位、12025位~12044位、12170位~12188位、12214位~12233位、12316位~12335位、12401位~12420位、12589位~12608位、12843位~12862位、12900位~12919位、12969位~12988位、13153位~13172位、13271位~13290位、13366位~13385位、13462位~13481位、13481位~13500位、13642位~13661位、13672位~13691位、13767位~13786位、13894位~13913位、14050位~14069位、14292位~14311位、14512位~14531位、14656位~14675位、14668位~14687位、14698位~14717位、14753位~14772位、14826位~14845位、14854位~14873位、14879位~14898位、14889位~14908位、14962位~14981位、14971位~14990位、14992位~15011位、15007位~15026位、15040位~15059位、15064位~15083位、15091位~15107位、15092位~15111位、15120位~15139位、15172位~15191位、15173位~15192位、15174位~15193位、15175位~15194位、15179位~15198位、15280位~15299位、15280位~15296位、15457位~15476位、15496位~15515位、15497位~15516位、15498位~15517位、15499位~15518位、15520位~15539位、15623位~15642位、15791位~15809位、15834位~15853位、15886位~15905位、15931位~15950位、15988位~16007位、16052位~16071位、16066位~16085位、16189位~16208位、16192位~16211位、16193位~16212位、16194位~16213位、16195位~16214位、16196位~16215位、16198位~16217位、16201位~16220位、16432位~16451位、16636位~16655位、16825位~16844位、17016位~17035位、17032位~17051位、17080位~17099位、17083位~17102位、17084位~17103位、17137位~17156位、17215位~17234位、17257位~17276位、17564位~17583位、17565位~17584位、17566位~17585位、17567位~17586位、17569位~17588位、17573位~17592位、17576位~17595位、17580位~17599位、17748位~17765位、17773位~17792位、17830位~17849位、17859位~17876位、18094位~18111位、18197位~18216位、18257位~18276位、18370位~18387位、19572位~19591位、19622位~19639位、19780位~19799位、19783位~19802位、20107位~20126位、20108位~20127位、20109位~20128位、20110位~20129位、20111位~20130位、20776位~20795位、20797位~20816位、20889位~20908位、21453位~21472位、21502位~21521位、21503位~21522位、21504位~21523位、22550位~22569位、22817位~22836位、23093位~23112位、23957位~23976位、24303位~24322位、24446位~24465位、24469位~24488位、24620位~24639位、24632位~24651位、24665位~24684位、24962位~24981位、25107位~25126位、25365位~25384位、25502位~25520位、26287位~26306位、26305位~26324位、26579位~26598位、26782位~26800位、27093位~27111位、27774位~27793位、27806位~27823位、28274位~28293位、28398位~28417位、28514位~28533位、28744位~28763位、28745位~28764位、28746位~28765位、28747位~28766位、28748位~28767位、28752位~28771位、28756位~28775位、28757位~28776位、28758位~28777位、28759位~28778位、28762位~28781位、28765位~28784位、28799位~28818位、28950位~28969位、28986位~29005位、29010位~29029位、29106位~29125位、29176位~29195位、29354位~29373位、29615位~29634位、29712位~29731位、及び29789位~29808位からなる群から選択される少なくとも一つの標的領域中の少なくとも10の連続する塩基を含む核酸に相補的である、(1)~(6)のいずれかに記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(8)配列番号1の704位~723位、5384位~5403位、6407位~6426位、10409位~10428位、10487位~10506位、12170位~12188位、12214位~12233位、12316位~12335位、12401位~12420位、13642位~13661位、13672位~13691位、13767位~13786位、13894位~13913位、14050位~14069位、14512位~14531位、14656位~14675位、14668位~14687位、14753位~14772位、14826位~14845位、14879位~14898位、14889位~14908位、14962位~14981位、14992位~15011位、15007位~15026位、15040位~15059位、15064位~15083位、15091位~15107位、15092位~15111位、15120位~15139位、15173位~15192位、15175位~15194位、15179位~15198位、15280位~15299位、15280位~15296位、15457位~15476位、15496位~15515位、15498位~15517位、15791位~15809位、15834位~15853位、15886位~15905位、15931位~15950位、15988位~16007位、16052位~16071位、16066位~16085位、16189位~16208位、16192位~16211位、16195位~16214位、16201位~16220位、16432位~16451位、16636位~16655位、17016位~17035位、17032位~17051位、17083位~17102位、17084位~17103位、17257位~17276位、17565位~17584位、17576位~17595位、17580位~17599位、17748位~17765位、17773位~17792位、17830位~17849位、18094位~18111位、18197位~18216位、18257位~18276位、19622位~19639位、19783位~19802位、20108位~20127位、20111位~20130位、20776位~20795位、20797位~20816位、20889位~20908位、21502位~21521位、21504位~21523位、24469位~24488位、24620位~24639位、24632位~24651位、24665位~24684位、24962位~24981位、25107位~25126位、26782位~26800位、27806位~27823位、28274位~28293位、28514位~28533位、28745位~28764位、28746位~28765位、28756位~28775位、28757位~28776位、28758位~28777位、28759位~28778位、28765位~28784位、28799位~28818位、28950位~28969位、29010位~29029位、29106位~29125位、29176位~29195位、29354位~29373位、及び29615位~29634位、及び29712位~29731位からなる群から選択される少なくとも一つの標的領域中の少なくとも10の連続する塩基を含む核酸に相補的である、(1)~(7)のいずれかに記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(9)配列番号1の5384位~5403位、6407位~6426位、13642位~13661位、13672位~13691位、15173位~15192位、15175位~15194位、15179位~15198位、15496位~15515位、15498位~15517位、16189位~16208位、16192位~16211位、16195位~16214位、16201位~16220位、17083位~17102位、17084位~17103位、17565位~17584位、17576位~17595位、18257位~18276位、19783位~19802位、20108位~20127位、20111位~20130位、21502位~21521位、21504位~21523位、24665位~24684位、28745位~28764位、28746位~28765位、28756位~28775位、28757位~28776位、28758位~28777位、及び28759位~28778位からなる群から選択される少なくとも一つの標的領域中の少なくとも10の連続する塩基を含む核酸に相補的である、(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(10)配列番号1の6407~6426位、15173位~15192位、15496~15515位、15498位~15517位、16195~16214位、17084位~17103位、20111位~20130位、21502~21521位、28757位~28776位、及び28758~28777位からなる群から選択される少なくとも一つの標的領域中の少なくとも10の連続する塩基を含む核酸に相補的である、(1)~(9)のいずれかに記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(11)標的領域中の少なくとも15の連続する塩基を含む核酸に相補的である、(1)~(10)のいずれかに記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(12)(a)配列番号2~177からなる群から選択される塩基配列、
(b)配列番号2~177からなる群から選択される塩基配列において1又は数個の塩基が付加、欠失、又は置換された塩基配列、又は
(c)配列番号2~177からなる群から選択される塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、前記標的領域の機能を阻害する、(1)~(11)のいずれかに記載のアンチセンスオリゴヌクレオチドもしくはその医薬的に許容可能な塩又はそれらの水和物。
(13)(a)配列番号7、9、12、19、20、23~26、36~40、42~44、46、47、49~51、53~59、61、63~68、70、74~82、85、88~90、92、93、95、96、99、101、106~110、112~114、117、119、121、124、125~127、129、131、138~143、149、152、153、155、157、158、162~165、167~169、及び171~176からなる群から選択される塩基配列、
(b)配列番号7、9、12、19、20、23~26、36~40、42~44、46、47、49~51、53~59、61、63~68、70、74~82、85、88~90、92、93、95、96、99、101、106~110、112~114、117、119、121、124、125~127、129、131、138~143、149、152、153、155、157、158、162~165、167~169、及び171~176からなる群から選択される塩基配列において1又は数個の塩基が付加、欠失、又は置換された塩基配列、又は
(c)配列番号7、9、12、19、20、23~26、36~40、42~44、46、47、49~51、53~59、61、63~68、70、74~82、85、88~90、92、93、95、96、99、101、106~110、112~114、117、119、121、124、125~127、129、131、138~143、149、152、153、155、157、158、162~165、167~169、及び171~176からなる群から選択される塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、前記標的領域の機能を阻害する、(1)~(12)のいずれかに記載のアンチセンスオリゴヌクレオチドもしくはその医薬的に許容可能な塩又はそれらの水和物。
(14)(a)配列番号9、12、36、37、61、63、64、68、70、81、82、85、88、95、96、101、106、114、119、121、124、129、131、141、157、158、162、163、164、及び165からなる群から選択される塩基配列、
(b)配列番号9、12、36、37、61、63、64、68、70、81、82、85、88、95、96、101、106、114、119、121、124、129、131、141、157、158、162、163、164、及び165からなる群から選択される塩基配列において1又は数個の塩基が付加、欠失、又は置換された塩基配列、又は
(c)配列番号9、12、36、37、61、63、64、68、70、81、82、85、88、95、96、101、106、114、119、121、124、129、131、141、157、158、162、163、164、及び165からなる群から選択される塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、前記標的領域の機能を阻害する、(1)~(13)のいずれかに記載のアンチセンスオリゴヌクレオチドもしくはその医薬的に許容可能な塩又はそれらの水和物。
(15)(a)配列番号12、61、68、70、85、96、124、129、163、及び164からなる群から選択される塩基配列、
(b)配列番号12、61、68、70、85、96、124、129、163、及び164からなる群から選択される塩基配列において1又は数個の塩基が付加、欠失、又は置換された塩基配列、又は
(c)配列番号12、61、68、70、85、96、124、129、163、及び164からなる群から選択される塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、前記標的領域の機能を阻害する、(1)~(14)のいずれかに記載のアンチセンスオリゴヌクレオチドもしくはその医薬的に許容可能な塩又はそれらの水和物。
(16)(a)の配列を含む、(11)~(15)のいずれかに記載のアンチセンスオリゴヌクレオチドもしくはその医薬的に許容可能な塩又はそれらの水和物。
(17-1)アンチセンスオリゴヌクレオチドが20ヌクレオチドからなる、(1)~(16)のいずれかに記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(17-2)アンチセンスオリゴヌクレオチドが、5'側から3'側にむかって、ヌクレオチド長5のウイング領域、ヌクレオチド長10のギャップ領域、及びヌクレオチド長10のウイング領域で構成されているギャップマーである、(17-1)に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(18)ウイング領域が2'-OMe(2'-O-CH3)基及び/又は2'-O-MOE(2'-O-CH2CH2OCH3)基を含む、(1)~(17-2)のいずれかに記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(19)5’ウイング領域の5'側から2番目と3番目のヌクレオシド間の結合および4番目と5番目のヌクレオシド間の結合がホスホジエステル結合であり、ならびに3’ウイング領域の5'側から1番目と2番目のヌクレオシド間の結合および3番目と4番目のヌクレオシド間の結合がホスホジエステル結合であり、かつその他のヌクレオシド間の結合が全てホスホロチオエート結合である、(17-1)、(17-2)、又は(18)に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(20)5’ウイング領域の5'側から2番目と3番目のヌクレオシド間の結合、3番目と4番目のヌクレオシド間の結合、および4番目と5番目のヌクレオシド間の結合がホスホジエステル結合であり、ならびに3’ウイング領域の5'側から1番目と2番目のヌクレオシド間の結合、2番目と3番目のヌクレオシド間の結合、および3番目と4番目のヌクレオシド間の結合がホスホジエステル結合であり、かつその他のヌクレオシド間の結合が全てホスホロチオエート結合である、(17-1)、(17-2)、又は(18)に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
(21)(1)~(20)のいずれかに記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物を含む、医薬組成物。
(22)SARS-CoV-2、SARS-CoV-1、及びMERS-CoVからなる群から選択されるウイルス感染症を治療及び/又は予防するための、(21)に記載の医薬組成物。
(23)(1)~(20)のいずれかに記載の核酸もしくはその医薬的に許容可能な塩又はそれらの水和物、又は(21)~(22)のいずれかに記載の医薬組成物の有効量を対象に投与する工程を含む、SARS-CoV-2、SARS-CoV-1、及びMERS-CoVからなる群から選択されるウイルス感染症の治療及び/又は予防方法。
(a)配列番号2~177からなる群から選択される塩基配列、
(b)配列番号2~177からなる群から選択される塩基配列において1又は数個の塩基が付加、欠失、又は置換された塩基配列、又は
(c)配列番号2~177からなる群から選択される塩基配列に対して、80%、85%以上、86%以上、87%以上、88%以上、89%以上、90%以上、91%以上、92%以上、93%以上、94%以上、95%以上、96%以上、97%以上、98%以上、又は99%以上の配列同一性を有する塩基配列
を含むか、又はこれらのいずれかの配列からなる。一実施形態において、本発明のアンチンセンスオリゴヌクレオチドは、上記(a)のいずれかの塩基配列を含むか、又はその配列からなる。
(a)配列番号7、9、12、19、20、23~26、36~40、42~44、46、47、49~51、53~59、61、63~68、70、74~82、85、88~90、92、93、95、96、99、101、106~110、112~114、117、119、121、124、125~127、129、131、138~143、149、152、153、155、157、158、162~165、167~169、及び171~176からなる群から選択される塩基配列、
(b)配列番号7、9、12、19、20、23~26、36~40、42~44、46、47、49~51、53~59、61、63~68、70、74~82、85、88~90、92、93、95、96、99、101、106~110、112~114、117、119、121、124、125~127、129、131、138~143、149、152、153、155、157、158、162~165、167~169、及び171~176からなる群から選択される塩基配列において1又は数個の塩基が付加、欠失、又は置換された塩基配列、又は
(c)配列番号7、9、12、19、20、23~26、36~40、42~44、46、47、49~51、53~59、61、63~68、70、74~82、85、88~90、92、93、95、96、99、101、106~110、112~114、117、119、121、124、125~127、129、131、138~143、149、152、153、155、157、158、162~165、167~169、及び171~176からなる群から選択される塩基配列に対して、80%以上、85%以上、86%以上、87%以上、88%以上、89%以上、90%以上、91%以上、92%以上、93%以上、94%以上、95%以上、96%以上、97%以上、98%以上、又は99%以上の配列同一性を有する塩基配列
を含むか、又はこれらのいずれかの配列からなる。一実施形態において、本発明のアンチンセンスオリゴヌクレオチドは、上記(a)のいずれかの塩基配列を含むか、又はその配列からなる。
(a)配列番号9、12、36、37、61、63、64、68、70、81、82、85、88、95、96、101、106、114、119、121、124、129、131、141、157、158、162、163、164、及び165からなる群から選択される塩基配列、
(b)配列番号9、12、36、37、61、63、64、68、70、81、82、85、88、95、96、101、106、114、119、121、124、129、131、141、157、158、162、163、164、及び165からなる群から選択される塩基配列において1又は数個の塩基が付加、欠失、又は置換された塩基配列、又は
(c)配列番号9、12、36、37、61、63、64、68、70、81、82、85、88、95、96、101、106、114、119、121、124、129、131、141、157、158、162、163、164、及び165からなる群から選択される塩基配列に対して、80%以上の配列同一性を有する塩基配列
を含むか、又はこれらのいずれかの配列からなる。一実施形態において、本発明のアンチンセンスオリゴヌクレオチドは、上記(a)のいずれかの塩基配列を含むか、又はその配列からなる。
一実施形態において、本発明のアンチンセンスオリゴヌクレオチドは、
(a)配列番号12、61、68、70、85、96、124、129、163、及び164からなる群から選択される塩基配列、
(b)配列番号12、61、68、70、85、96、124、129、163、及び164からなる群から選択される塩基配列において1又は数個の塩基が付加、欠失、又は置換された塩基配列、又は
(c)配列番号12、61、68、70、85、96、124、129、163、及び164からなる群から選択される塩基配列に対して、80%以上、85%以上、86%以上、87%以上、88%以上、89%以上、90%以上、91%以上、92%以上、93%以上、94%以上、95%以上、96%以上、97%以上、98%以上、又は99%以上の配列同一性を有する塩基配列
を含むか、又はこれらのいずれかの配列からなる。一実施形態において、本発明のアンチンセンスオリゴヌクレオチドは、上記(a)のいずれかの塩基配列を含むか、又はその配列からなる。
SARS-CoV-2(リファレンス配列; NC_045512.2)(配列番号1)のゲノムRNAとSARS-CoV-1(リファレンス配列; NC_004718.3)のゲノムRNAの配列を比較し、2種間で保存されている領域を特定した。2種間で保存されている領域として、配列番号1の43位~89位、242~279位、290~312位、455~477位、704~723位、3352位~3372位、5384~5403位、6071~6090位、6406位~6427位、6797~6815位、7532~7551位、8707~8724位、10292~10309位、10406位~10431位、10484位~10506位、11609~11630位、12023~12045位、12170~12188位、12212~12234位、12314~12339位、12401~12420位、12589~12608位、12839位~12867位、12898位~12922位、12965位~12990位、13151~13175位、13271~13290位、13363~13386位、13458位~13502位、13642~13661位、13672~13691位、13762位~13790位、13894位~13916位、14050~14069位、14290位~14312位、14512~14531位、14654位~14687位、14698~14717位、14750位~14777位、14824位~14846位、14854~14873位、14878位~14909位、14953位~14990位、14992位~15026位、15037位~15061位、15063位~15140位、15172位~15198位、15278位~15299位、15454位~15479位、15496位~15518位、15520~15539位、15622位~15644位、15791~15809位、15829位~15859位、15886~15905位、15929位~15950位、15985~16011位、16051位~16085位、16189位~16220位、16430位~16451位、16636~16655位、16822位~16845位、17015位~17051位、17080位~17103位、17137~17156位、17215~17234位、17254位~17277位、17564位~17600位、17748~17765位、17773~17792位、17830~17849位、17859~17876位、18094~18111位、18196位~18218位、18253位~18278位、18370~18387位、19568位~19595位、19622~19639位、19780~19802位、20107~20130位、20776~20795位、20797~20816位、20888位~20909位、21453~21472位、21502位~21524位、22550~22569位、22814位~22836位、23093位~23113位、23956位~23976位、24302位~24324位、24446~24465位、24467位~24489位、24620位~24651位、24662位~24684位、24962位~24982位、25104位~25128位、25364位~25384位、25502~25520位、26287位~26325位、26574位~26604位、26782~26800位、27093~27111位、27771~27794位、27806~27823位、28270位~28294位、28397位~28418位、28509位~28538位、28744位~28784位、28799位~28820位、28946位~28972位、28986~29005位、29010位~29031位、29102位~29130位、29174位~29196位、29354~29373位、29615~29634位、29712~29731位、及び29787~29867位の塩基配列を見出した。当該保存領域中に含まれる配列を標的として、被験物質gapmer No.G1~G176(配列番号2~177)を作製した。被験物質gapmer No.G1~G176はウイング領域全てのヌクレオシドが2’-OMe(2’-O-CH3)基を含む。また、実施例中、gamper No.の末尾に「MOE」が付されたgapmerは、2’ -O-MOE gapmer(ウイング部分の全てのヌクレオシドが2’-O-MOE(2'-O-CH2CH2OCH3)を含むギャップマー)である。また、実施例の表1-1において、ヌクレオシド間の結合は全てホスホロチオエート結合である。また、実施例において、オリゴヌクレオチドは、以下のオリゴヌクレオチドを除き、原則として全て5-10-5型のギャップマーである。
19mer 4-10-5型:G110、G114
19mer 5-10-4型:G11、G19、G61、G115
18mer 4-10-4型:G13、G14、G81、G84、G85、G88、G90
18mer 5-9-4型:G117
17mer 4-9-4型:133-2’OMe、133-2’MOE、134-2’OMe、134-2’MOE
なお、上記ギャップマーにおいて「a-b-c型」とは、ギャップマーが、5'側から3'側にむかって、ヌクレオチド長aのウイング領域、ヌクレオチド長bのギャップ領域、ヌクレオチド長cのウイング領域で構成されていることを意味する。
なお、2’-O-MOE gapmerでは、ウイング領域のCには2’-O-MOE-5-methyl Cを使用し、ギャップ領域のCには5-methyl dCを使用した。
標的配列及び被験物質gapmer No.G1~G176の配列等を以下の表1-1に示す。
また、標的配列及びPS/PS型の被験物質gapmer No.G58 PO/PS type-1、G58 PO/PS-type-2、G144 PO/PS type-1、PO/PS type-2の配列等を以下の表1-2に示す。表1-2の5-10-5型のギャップマーにおいて、5’ウイング領域の5'側から2番目と3番目のヌクレオシド間の結合および4番目と5番目のヌクレオシド間の結合がホスホジエステル結合であり、ならびに3’ウイング領域の5'側から1番目と2番目のヌクレオシド間の結合および3番目と4番目のヌクレオシド間の結合がホスホジエステル結合であり、かつその他のヌクレオシド間の結合が全てホスホロチオエート結合である(PO/PS型-2)か、5’ウイング領域の5'側から2番目と3番目のヌクレオシド間の結合、3番目と4番目のヌクレオシド間の結合、および4番目と5番目のヌクレオシド間の結合がホスホジエステル結合であり、ならびに3’ウイング領域の5'側から1番目と2番目のヌクレオシド間の結合、2番目と3番目のヌクレオシド間の結合、および3番目と4番目のヌクレオシド間の結合がホスホジエステル結合であり、かつその他のヌクレオシド間の結合が全てホスホロチオエート結合である(PO/PS型-1)。
終濃度2~10 μMの被験物質gapmer又は陰性対照gapmerを、293T細胞にヒトACE2発現レンチウイルスベクターを感染させて作製したヒトACE2安定発現細胞(293T-ACE2)にAmxa Cell Line Nucleofector Kit V(Lonza)(solution V 73.6uL及びsupplement 16.4uL/sample)とNucleofectorII(Amaxa)を使用し、NucleofectorIIに搭載されているパルスプログラムよりA-023を選択し、エレクトロポレーションを行った。陰性対照gapmerにはASO#129700 (Wheeler et al. 2012. Nature 488: 111-5. doi: 10.1038/nature11362)の配列を用いた。被験物質gapmerは、実施例1で用いたGapmer No. 1から135の内で一部未評価であった配列、及びGapmer No. 1から135の活性データを元に追加設計したGapmer No. 136からG176を用いた。被験物質gapmer及び陰性対照gapmerは2’-OMe gapmer又は2’-MOE gapmerとして、日本バイオサービス又は味の素バイオファーマサービスに合成を委託した。
293T-ACE2はエレクトロポレーション1回当たり6.0×105cellsを使用した。エレクトロポレーション後、96-wellプレートに293T-ACE2を5.0×104 cells/wellで播種した。翌日、SARS-CoV-2ウイルス液をMOI=0.01で添加し、細胞にウイルスを感染させた。SARS-CoV-2ウイルスは国立感染症研究所から入手したWK521株を元に、文献(Terada et al. 2019. J Virol 93: e01208-19. doi: 10.1128/JVI.01208-19)に記載の手法と同様の手法を用いてアクセサリータンパク質ORF8のコード領域にNano-luciferase遺伝子を組込んだrecombinant SARS-CoV-2ウイルス(rSARS-CoV-2-ORF8-Nluc)を用いた。ウイルス液添加から1h後に培地交換により培地中のウイルスを除去した。ウイルス感染から48h後に、培養上清を回収し、細胞はPassive Lysis Buffer (Promega)を用いて細胞溶解液を調製した。Luciferase Assay System (Promega)を用いて添付のプロトコルにしたがって細胞溶解液中のluciferase活性を測定し、細胞内のSARS-CoV-2ウイルス量を評価した(細胞内ウイルス量の測定)。また、回収した培養上清を、別途96-wellプレートに3.0×104 cells/wellで播種した293T-ACE2細胞に5μL/wellで添加し、培養上清中のウイルスを細胞に感染させた。培養上清による感染から24h後に、最初のウイルス感染と同様の方法で細胞内のSARS-CoV-2ウイルス量を評価し、培養上清中の感染性ウイルス量を評価した(培地中感染性ウイルス量の測定)。被験物質gapmerのノックダウン活性(培地中感染性ウイルス量抑制率:陰性対照gapemer導入時の感染性ウイルス量に対する、被験物質gapmer導入時の感染性ウイルス量抑制率)を表2に示す。
Claims (23)
- SARS-CoV-2のゲノムRNAにおける、5'UTR領域、nsp1領域、nsp3領域、nsp4領域、3C-like proteinase領域、nsp6領域、nsp7領域、nsp8領域、nsp9領域、nsp10領域、RNA-dependent RNA polymerase領域、helicase領域、3'-to-5' exonuclease領域、endoRNase領域、2'-O-ribose methyltransferase領域、S1領域及びS2領域を含むS領域、ORF3a領域、E領域、M領域、ORF7b領域、N領域、ORF10領域、及び3'UTR領域からなる群から選択される少なくとも一つの標的領域中の少なくとも10の連続する塩基を含む核酸に相補的である、15~30ヌクレオチドからなるアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物であって、
SARS-CoV-2、SARS-CoV-1、及びMERS-CoVからなる群から選択されるウイルスに対する抗ウイルス効果を有する、アンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。 - アンチセンスオリゴヌクレオチドが中央のギャップ領域、及びギャップ領域に隣接する2つのウイング領域を含むギャップマーである、請求項1に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- ウイルスがSARS-CoV-2又はSARS-CoV-1である、請求項1又は2に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- 少なくとも一つの標的領域が、SARS-CoV-2のゲノムRNAにおける、nsp1領域、nsp3領域、3C-like proteinase領域、nsp8領域、RNA-dependent RNA polymerase領域、helicase領域、3'-to-5' exonuclease領域、endoRNase領域、2'-O-ribose methyltransferase領域、S1領域及びS2領域を含むS領域、M領域、ORF7b領域、N領域、ORF10領域、及び3'UTR領域からなる群から選択される、請求項1~3のいずれか一項に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- 少なくとも一つの標的領域が、SARS-CoV-2のゲノムRNAにおける、nsp3領域、RNA-dependent RNA polymerase領域、helicase領域、3'-to-5' exonuclease領域、endoRNase領域、2'-O-ribose methyltransferase領域、S1領域及びS2領域を含むS領域、及びN領域からなる群から選択される、請求項1~4のいずれか一項に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- 配列番号1の43位~89位、242~279位、290~312位、455~477位、704~723位、3352位~3372位、5384~5403位、6071~6090位、6406位~6427位、6797~6815位、7532~7551位、8707~8724位、10292~10309位、10406位~10431位、10484位~10506位、11609~11630位、12023~12045位、12170~12188位、12212~12234位、12314~12339位、12401~12420位、12589~12608位、12839位~12867位、12898位~12922位、12965位~12990位、13151~13175位、13271~13290位、13363~13386位、13458位~13502位、13642~13661位、13672~13691位、13762位~13790位、13894位~13916位、14050~14069位、14290位~14312位、14512~14531位、14654位~14687位、14698~14717位、14750位~14777位、14824位~14846位、14854~14873位、14878位~14909位、14953位~14990位、14992位~15026位、15037位~15061位、15063位~15140位、15172位~15198位、15278位~15299位、15454位~15479位、15496位~15518位、15520~15539位、15622位~15644位、15791~15809位、15829位~15859位、15886~15905位、15929位~15950位、15985~16011位、16051位~16085位、16189位~16220位、16430位~16451位、16636~16655位、16822位~16845位、17015位~17051位、17080位~17103位、17137~17156位、17215~17234位、17254位~17277位、17564位~17600位、17748~17765位、17773~17792位、17830~17849位、17859~17876位、18094~18111位、18196位~18218位、18253位~18278位、18370~18387位、19568位~19595位、19622~19639位、19780~19802位、20107~20130位、20776~20795位、20797~20816位、20888位~20909位、21453~21472位、21502位~21524位、22550~22569位、22814位~22836位、23093位~23113位、23956位~23976位、24302位~24324位、24446~24465位、24467位~24489位、24620位~24651位、24662位~24684位、24962位~24982位、25104位~25128位、25364位~25384位、25502~25520位、26287位~26325位、26574位~26604位、26782~26800位、27093~27111位、27771~27794位、27806~27823位、28270位~28294位、28397位~28418位、28509位~28538位、28744位~28784位、28799位~28820位、28946位~28972位、28986~29005位、29010位~29031位、29102位~29130位、29174位~29196位、29354~29373位、29615~29634位、29712~29731位、及び29787~29867位からなる群から選択される少なくとも一つの標的領域中の少なくとも10の連続する塩基を含む核酸に相補的である、請求項1~5のいずれか一項に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- 配列番号1の47位~66位、242位~261位、259位~278位、292位~311位、456位~475位、704位~723位、3353位~3372位、5384位~5403位、6071位~6090位、6406位~6425位、6407位~6426位、6408位~6427位、6797位~6815位、7532位~7551位、8707位~8724位、10292位~10309位、10407位~10426位、10409位~10428位、10487位~10506位、11609位~11628位、12025位~12044位、12170位~12188位、12214位~12233位、12316位~12335位、12401位~12420位、12589位~12608位、12843位~12862位、12900位~12919位、12969位~12988位、13153位~13172位、13271位~13290位、13366位~13385位、13462位~13481位、13481位~13500位、13642位~13661位、13672位~13691位、13767位~13786位、13894位~13913位、14050位~14069位、14292位~14311位、14512位~14531位、14656位~14675位、14668位~14687位、14698位~14717位、14753位~14772位、14826位~14845位、14854位~14873位、14879位~14898位、14889位~14908位、14962位~14981位、14971位~14990位、14992位~15011位、15007位~15026位、15040位~15059位、15064位~15083位、15091位~15107位、15092位~15111位、15120位~15139位、15172位~15191位、15173位~15192位、15174位~15193位、15175位~15194位、15179位~15198位、15280位~15299位、15280位~15296位、15457位~15476位、15496位~15515位、15497位~15516位、15498位~15517位、15499位~15518位、15520位~15539位、15623位~15642位、15791位~15809位、15834位~15853位、15886位~15905位、15931位~15950位、15988位~16007位、16052位~16071位、16066位~16085位、16189位~16208位、16192位~16211位、16193位~16212位、16194位~16213位、16195位~16214位、16196位~16215位、16198位~16217位、16201位~16220位、16432位~16451位、16636位~16655位、16825位~16844位、17016位~17035位、17032位~17051位、17080位~17099位、17083位~17102位、17084位~17103位、17137位~17156位、17215位~17234位、17257位~17276位、17564位~17583位、17565位~17584位、17566位~17585位、17567位~17586位、17569位~17588位、17573位~17592位、17576位~17595位、17580位~17599位、17748位~17765位、17773位~17792位、17830位~17849位、17859位~17876位、18094位~18111位、18197位~18216位、18257位~18276位、18370位~18387位、19572位~19591位、19622位~19639位、19780位~19799位、19783位~19802位、20107位~20126位、20108位~20127位、20109位~20128位、20110位~20129位、20111位~20130位、20776位~20795位、20797位~20816位、20889位~20908位、21453位~21472位、21502位~21521位、21503位~21522位、21504位~21523位、22550位~22569位、22817位~22836位、23093位~23112位、23957位~23976位、24303位~24322位、24446位~24465位、24469位~24488位、24620位~24639位、24632位~24651位、24665位~24684位、24962位~24981位、25107位~25126位、25365位~25384位、25502位~25520位、26287位~26306位、26305位~26324位、26579位~26598位、26782位~26800位、27093位~27111位、27774位~27793位、27806位~27823位、28274位~28293位、28398位~28417位、28514位~28533位、28744位~28763位、28745位~28764位、28746位~28765位、28747位~28766位、28748位~28767位、28752位~28771位、28756位~28775位、28757位~28776位、28758位~28777位、28759位~28778位、28762位~28781位、28765位~28784位、28799位~28818位、28950位~28969位、28986位~29005位、29010位~29029位、29106位~29125位、29176位~29195位、29354位~29373位、29615位~29634位、29712位~29731位、及び29789位~29808位からなる群から選択される少なくとも一つの標的領域中の少なくとも10の連続する塩基を含む核酸に相補的である、請求項1~6のいずれか一項に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- 配列番号1の704位~723位、5384位~5403位、6407位~6426位、10409位~10428位、10487位~10506位、12170位~12188位、12214位~12233位、12316位~12335位、12401位~12420位、13642位~13661位、13672位~13691位、13767位~13786位、13894位~13913位、14050位~14069位、14512位~14531位、14656位~14675位、14668位~14687位、14753位~14772位、14826位~14845位、14879位~14898位、14889位~14908位、14962位~14981位、14992位~15011位、15007位~15026位、15040位~15059位、15064位~15083位、15091位~15107位、15092位~15111位、15120位~15139位、15173位~15192位、15175位~15194位、15179位~15198位、15280位~15299位、15280位~15296位、15457位~15476位、15496位~15515位、15498位~15517位、15791位~15809位、15834位~15853位、15886位~15905位、15931位~15950位、15988位~16007位、16052位~16071位、16066位~16085位、16189位~16208位、16192位~16211位、16195位~16214位、16201位~16220位、16432位~16451位、16636位~16655位、17016位~17035位、17032位~17051位、17083位~17102位、17084位~17103位、17257位~17276位、17565位~17584位、17576位~17595位、17580位~17599位、17748位~17765位、17773位~17792位、17830位~17849位、18094位~18111位、18197位~18216位、18257位~18276位、19622位~19639位、19783位~19802位、20108位~20127位、20111位~20130位、20776位~20795位、20797位~20816位、20889位~20908位、21502位~21521位、21504位~21523位、24469位~24488位、24620位~24639位、24632位~24651位、24665位~24684位、24962位~24981位、25107位~25126位、26782位~26800位、27806位~27823位、28274位~28293位、28514位~28533位、28745位~28764位、28746位~28765位、28756位~28775位、28757位~28776位、28758位~28777位、28759位~28778位、28765位~28784位、28799位~28818位、28950位~28969位、29010位~29029位、29106位~29125位、29176位~29195位、29354位~29373位、及び29615位~29634位、及び29712位~29731位からなる群から選択される少なくとも一つの標的領域中の少なくとも10の連続する塩基を含む核酸に相補的である、請求項1~7のいずれか一項に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- 配列番号1の5384位~5403位、6407位~6426位、13642位~13661位、13672位~13691位、15173位~15192位、15175位~15194位、15179位~15198位、15496位~15515位、15498位~15517位、16189位~16208位、16192位~16211位、16195位~16214位、16201位~16220位、17083位~17102位、17084位~17103位、17565位~17584位、17576位~17595位、18257位~18276位、19783位~19802位、20108位~20127位、20111位~20130位、21502位~21521位、21504位~21523位、24665位~24684位、28745位~28764位、28746位~28765位、28756位~28775位、28757位~28776位、28758位~28777位、及び28759位~28778位からなる群から選択される少なくとも一つの標的領域中の少なくとも10の連続する塩基を含む核酸に相補的である、請求項1~8のいずれか一項に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- 配列番号1の6407~6426位、15173位~15192位、15496~15515位、15498位~15517位、16195~16214位、17084位~17103位、20111位~20130位、21502~21521位、28757位~28776位、及び28758~28777位からなる群から選択される少なくとも一つの標的領域中の少なくとも10の連続する塩基を含む核酸に相補的である、請求項1~9のいずれか一項に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- 標的領域中の少なくとも15の連続する塩基を含む核酸に相補的である、請求項1~10のいずれか一項に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- (a)配列番号2~177からなる群から選択される塩基配列、
(b)配列番号2~177からなる群から選択される塩基配列において1又は数個の塩基が付加、欠失、又は置換された塩基配列、又は
(c)配列番号2~177からなる群から選択される塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、前記標的領域の機能を阻害する、請求項1~11のいずれか一項に記載のアンチセンスオリゴヌクレオチドもしくはその医薬的に許容可能な塩又はそれらの水和物。 - (a)配列番号7、9、12、19、20、23~26、36~40、42~44、46、47、49~51、53~59、61、63~68、70、74~82、85、88~90、92、93、95、96、99、101、106~110、112~114、117、119、121、124、125~127、129、131、138~143、149、152、153、155、157、158、162~165、167~169、及び171~176からなる群から選択される塩基配列、
(b)配列番号7、9、12、19、20、23~26、36~40、42~44、46、47、49~51、53~59、61、63~68、70、74~82、85、88~90、92、93、95、96、99、101、106~110、112~114、117、119、121、124、125~127、129、131、138~143、149、152、153、155、157、158、162~165、167~169、及び171~176からなる群から選択される塩基配列において1又は数個の塩基が付加、欠失、又は置換された塩基配列、又は
(c)配列番号7、9、12、19、20、23~26、36~40、42~44、46、47、49~51、53~59、61、63~68、70、74~82、85、88~90、92、93、95、96、99、101、106~110、112~114、117、119、121、124、125~127、129、131、138~143、149、152、153、155、157、158、162~165、167~169、及び171~176からなる群から選択される塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、前記標的領域の機能を阻害する、請求項1~12のいずれか一項に記載のアンチセンスオリゴヌクレオチドもしくはその医薬的に許容可能な塩又はそれらの水和物。 - (a)配列番号9、12、36、37、61、63、64、68、70、81、82、85、88、95、96、101、106、114、119、121、124、129、131、141、157、158、162、163、164、及び165からなる群から選択される塩基配列、
(b)配列番号9、12、36、37、61、63、64、68、70、81、82、85、88、95、96、101、106、114、119、121、124、129、131、141、157、158、162、163、164、及び165からなる群から選択される塩基配列において1又は数個の塩基が付加、欠失、又は置換された塩基配列、又は
(c)配列番号9、12、36、37、61、63、64、68、70、81、82、85、88、95、96、101、106、114、119、121、124、129、131、141、157、158、162、163、164、及び165からなる群から選択される塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、前記標的領域の機能を阻害する、請求項1~13のいずれか一項に記載のアンチセンスオリゴヌクレオチドもしくはその医薬的に許容可能な塩又はそれらの水和物。 - (a)配列番号12、61、68、70、85、96、124、129、163、及び164からなる群から選択される塩基配列、
(b)配列番号12、61、68、70、85、96、124、129、163、及び164からなる群から選択される塩基配列において1又は数個の塩基が付加、欠失、又は置換された塩基配列、又は
(c)配列番号12、61、68、70、85、96、124、129、163、及び164からなる群から選択される塩基配列に対して、80%以上の配列同一性を有する塩基配列を含み、前記標的領域の機能を阻害する、請求項1~14のいずれか一項に記載のアンチセンスオリゴヌクレオチドもしくはその医薬的に許容可能な塩又はそれらの水和物。 - (a)の配列を含む、請求項11~15のいずれか一項に記載のアンチセンスオリゴヌクレオチドもしくはその医薬的に許容可能な塩又はそれらの水和物。
- アンチセンスオリゴヌクレオチドが20ヌクレオチドからなる、請求項1~16のいずれか一項に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- ウイング領域が2'-OMe(2'-O-CH3)基及び/又は2'-O-MOE(2'-O-CH2CH2OCH3)基を含む、請求項1~17のいずれか一項に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- 5’ウイング領域の5'側から2番目と3番目のヌクレオシド間の結合および4番目と5番目のヌクレオシド間の結合がホスホジエステル結合であり、ならびに3’ウイング領域の5'側から1番目と2番目のヌクレオシド間の結合および3番目と4番目のヌクレオシド間の結合がホスホジエステル結合であり、かつその他のヌクレオシド間の結合が全てホスホロチオエート結合である、請求項17又は18に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- 5’ウイング領域の5'側から2番目と3番目のヌクレオシド間の結合、3番目と4番目のヌクレオシド間の結合、および4番目と5番目のヌクレオシド間の結合がホスホジエステル結合であり、ならびに3’ウイング領域の5'側から1番目と2番目のヌクレオシド間の結合、2番目と3番目のヌクレオシド間の結合、および3番目と4番目のヌクレオシド間の結合がホスホジエステル結合であり、かつその他のヌクレオシド間の結合が全てホスホロチオエート結合である、請求項17又は18に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物。
- 請求項1~20のいずれか一項に記載のアンチセンスオリゴヌクレオチド又はその医薬的に許容可能な塩もしくはそれらの水和物を含む、医薬組成物。
- SARS-CoV-2、SARS-CoV-1、及びMERS-CoVからなる群から選択されるウイルス感染症を治療及び/又は予防するための、請求項21に記載の医薬組成物。
- 請求項1~19のいずれか一項に記載の核酸もしくはその医薬的に許容可能な塩又はそれらの水和物、又は請求項21~22のいずれか一項に記載の医薬組成物の有効量を対象に投与する工程を含む、SARS-CoV-2、SARS-CoV-1、及びMERS-CoVからなる群から選択されるウイルス感染症の治療及び/又は予防方法。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202280072073.4A CN118265787A (zh) | 2021-10-26 | 2022-10-26 | 抗病毒反义寡核苷酸 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2021174879 | 2021-10-26 | ||
JP2021-174879 | 2021-10-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2023074748A1 true WO2023074748A1 (ja) | 2023-05-04 |
Family
ID=86158036
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2022/039960 WO2023074748A1 (ja) | 2021-10-26 | 2022-10-26 | 抗ウイルスアンチセンスオリゴヌクレオチド |
Country Status (3)
Country | Link |
---|---|
CN (1) | CN118265787A (ja) |
TW (1) | TW202334417A (ja) |
WO (1) | WO2023074748A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023171804A1 (ja) * | 2022-03-10 | 2023-09-14 | 日本新薬株式会社 | 抗ウイルスアンチセンスオリゴマー |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2924179B2 (ja) | 1993-02-19 | 1999-07-26 | 日本新薬株式会社 | グリセロール誘導体,デバイス及び医薬組成物 |
US20210317457A1 (en) * | 2020-04-10 | 2021-10-14 | Aligos Therapeutics, Inc. | Antisense oligonucleotide (aso) molecules and uses thereof for coronavirus diseases |
-
2022
- 2022-10-26 WO PCT/JP2022/039960 patent/WO2023074748A1/ja active Application Filing
- 2022-10-26 TW TW111140728A patent/TW202334417A/zh unknown
- 2022-10-26 CN CN202280072073.4A patent/CN118265787A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2924179B2 (ja) | 1993-02-19 | 1999-07-26 | 日本新薬株式会社 | グリセロール誘導体,デバイス及び医薬組成物 |
US20210317457A1 (en) * | 2020-04-10 | 2021-10-14 | Aligos Therapeutics, Inc. | Antisense oligonucleotide (aso) molecules and uses thereof for coronavirus diseases |
Non-Patent Citations (12)
Title |
---|
"World Health Organization (WHO", 11 March 2020, PRESS RELEASE |
ALTSCHUL SF ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 |
BALDASSARRE ANTONELLA, PAOLINI ALESSANDRO, BRUNO STEFANIA PAOLA, FELLI CRISTINA, TOZZI ALBERTO EUGENIO, MASOTTI ANDREA: "Potential use of noncoding RNAs and innovative therapeutic strategies to target the 5’UTR of SARS-CoV-2", EPIGENOMICS, vol. 12, no. 15, 1 August 2020 (2020-08-01), United Kingdom , pages 1349 - 1361, XP055804747, ISSN: 1750-1911, DOI: 10.2217/epi-2020-0162 * |
BIJSTERBOSCH, M.K. ET AL., NUCLEIC ACID RES., vol. 28, 2000, pages 2717 - 2725 |
ENYA ET AL., BIOORGANIC & MEDICINAL CHEMISTRY, vol. 18, 2008, pages 9154 - 9160 |
KARLINALTSCHUL: "Basic Local Alignment Search Tool", PROC. NATL. ACAD. SCI. USA, 1990, pages 872264 - 2268 |
LULLA VALERIA, WANDEL MICHAL P, BANDYRA KATARZYNA J, ULFERTS RACHEL, WU MARY, DENDOOVEN TOM, YANG XIAOFEI, DOYLE NICOLE, OERUM STE: "Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome", JOURNAL OF VIROLOGY, 24 June 2021 (2021-06-24), XP055886910, DOI: 10.1128/JVI.00663-21 * |
NAT MICROBIOL., vol. 5, no. 4, April 2020 (2020-04-01), pages 536 - 544 |
PROC NATL ACAD SCI USA, vol. 90, 1993, pages 5873 |
TERADA ET AL., J VIROL, vol. 93, 2019, pages e01208 - 19 |
W. BRAD WAN ET AL., NUCLEIC ACID RESEARCH, vol. 42, no. 22, 2014, pages 13456 |
WHEELER ET AL., NATURE, vol. 488, 2012, pages 111 - 5 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023171804A1 (ja) * | 2022-03-10 | 2023-09-14 | 日本新薬株式会社 | 抗ウイルスアンチセンスオリゴマー |
Also Published As
Publication number | Publication date |
---|---|
TW202334417A (zh) | 2023-09-01 |
CN118265787A (zh) | 2024-06-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11851658B2 (en) | Methods and compositions for the specific inhibition of alpha-1 antitrypsin by double-stranded RNA | |
EP3052632A1 (en) | Compositions and methods for treating amyotrophic lateral sclerosis | |
CN101103111A (zh) | 用于治疗呼吸道病毒感染的组合物及其用途 | |
US9850486B2 (en) | Methods and compositions for the specific inhibition of CKAP5 by double-stranded RNA | |
WO2006038608A1 (ja) | オリゴ二本鎖rna及び医薬組成物 | |
WO2023074748A1 (ja) | 抗ウイルスアンチセンスオリゴヌクレオチド | |
ES2909308T3 (es) | Métodos para modular la expresión de MECP2 | |
US9574243B2 (en) | Compositions and methods for the treatment of influenza infection | |
CN112534055A (zh) | 用于调节rtel1表达的寡核苷酸 | |
EP4332223A1 (en) | Antiviral nucleic acid | |
US20240229035A1 (en) | Antiviral nucleic acid | |
JP2023161422A (ja) | 二本鎖領域を有する抗ウイルス核酸 | |
TW202346589A (zh) | 抗病毒反義寡聚物 | |
WO2023112931A1 (ja) | ATN1 mRNA又はpre-mRNAを標的とするアンチセンスオリゴヌクレオチド | |
WO2022270585A1 (ja) | アンチセンスオリゴマーの組み合わせ | |
KR20230030690A (ko) | 코로나바이러스감염증-19의 치료에 사용하기 위한 SARS-CoV-2 번역 조절 저해 활성을 갖는 안티센스 올리고뉴클레오티드 | |
WO2007052629A1 (ja) | オリゴ二本鎖rna及び医薬組成物 | |
JP2023506547A (ja) | B型肝炎ウイルス感染を処置するためのcops3阻害剤の使用 | |
JP2023506954A (ja) | B型肝炎ウイルス感染を処置するためのsaraf阻害剤の使用 | |
CN102618541A (zh) | 用于治疗呼吸道病毒感染的组合物及其用途 |
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: 22887073 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2023556602 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2022887073 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2022887073 Country of ref document: EP Effective date: 20240527 |