US20170283496A1 - Oligonucleotides for reduction of pd-l1 expression - Google Patents
Oligonucleotides for reduction of pd-l1 expression Download PDFInfo
- Publication number
- US20170283496A1 US20170283496A1 US15/458,800 US201715458800A US2017283496A1 US 20170283496 A1 US20170283496 A1 US 20170283496A1 US 201715458800 A US201715458800 A US 201715458800A US 2017283496 A1 US2017283496 A1 US 2017283496A1
- Authority
- US
- United States
- Prior art keywords
- oligonucleotide
- nucleosides
- region
- conjugate
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108091034117 Oligonucleotide Proteins 0.000 title claims abstract description 646
- 230000014509 gene expression Effects 0.000 title claims abstract description 80
- 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 title abstract description 114
- 230000009467 reduction Effects 0.000 title description 19
- 108010074708 B7-H1 Antigen Proteins 0.000 claims abstract description 203
- 102000008096 B7-H1 Antigen Human genes 0.000 claims abstract description 201
- 210000004185 liver Anatomy 0.000 claims abstract description 98
- 239000000074 antisense oligonucleotide Substances 0.000 claims abstract description 71
- 238000012230 antisense oligonucleotides Methods 0.000 claims abstract description 71
- 238000000034 method Methods 0.000 claims abstract description 68
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 38
- 208000015181 infectious disease Diseases 0.000 claims abstract description 23
- 230000003612 virological effect Effects 0.000 claims abstract description 19
- 208000030852 Parasitic disease Diseases 0.000 claims abstract description 15
- 230000036281 parasite infection Effects 0.000 claims abstract description 13
- 201000005485 Toxoplasmosis Diseases 0.000 claims abstract description 9
- 201000004792 malaria Diseases 0.000 claims abstract description 9
- 208000004554 Leishmaniasis Diseases 0.000 claims abstract description 8
- 201000002311 trypanosomiasis Diseases 0.000 claims abstract description 8
- 206010027457 Metastases to liver Diseases 0.000 claims abstract description 6
- 201000007270 liver cancer Diseases 0.000 claims abstract description 6
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 6
- 125000003835 nucleoside group Chemical group 0.000 claims description 323
- 239000002777 nucleoside Substances 0.000 claims description 291
- 125000003729 nucleotide group Chemical group 0.000 claims description 172
- 239000002773 nucleotide Substances 0.000 claims description 171
- 210000004027 cell Anatomy 0.000 claims description 149
- 150000007523 nucleic acids Chemical class 0.000 claims description 140
- 108020004707 nucleic acids Proteins 0.000 claims description 139
- 102000039446 nucleic acids Human genes 0.000 claims description 139
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 84
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 claims description 64
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical group CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 claims description 62
- 230000000295 complement effect Effects 0.000 claims description 61
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 45
- 230000008685 targeting Effects 0.000 claims description 44
- 102000005427 Asialoglycoprotein Receptor Human genes 0.000 claims description 40
- 108010006523 asialoglycoprotein receptor Proteins 0.000 claims description 40
- 201000010099 disease Diseases 0.000 claims description 35
- 150000004713 phosphodiesters Chemical class 0.000 claims description 35
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 claims description 27
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 claims description 27
- 238000000338 in vitro Methods 0.000 claims description 23
- 239000000427 antigen Substances 0.000 claims description 22
- 108091007433 antigens Proteins 0.000 claims description 22
- 102000036639 antigens Human genes 0.000 claims description 22
- 238000001727 in vivo Methods 0.000 claims description 20
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 claims description 13
- 241000700605 Viruses Species 0.000 claims description 12
- 230000028993 immune response Effects 0.000 claims description 12
- 239000003085 diluting agent Substances 0.000 claims description 10
- 244000045947 parasite Species 0.000 claims description 9
- 150000003839 salts Chemical class 0.000 claims description 8
- 239000002671 adjuvant Substances 0.000 claims description 7
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 6
- 229930182830 galactose Natural products 0.000 claims description 5
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 4
- MSWZFWKMSRAUBD-GASJEMHNSA-N 2-amino-2-deoxy-D-galactopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 claims description 3
- KXTUJUVCAGXOBN-WQXQQRIOSA-N 2-methyl-N-[(3R,4R,5R,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)oxan-3-yl]propanamide Chemical compound CC(C)C(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O KXTUJUVCAGXOBN-WQXQQRIOSA-N 0.000 claims description 3
- RPJMPMDUKSRLLF-QNRYFBKSSA-N N-[(3R,4R,5R,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)oxan-3-yl]butanamide Chemical compound CCCC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O RPJMPMDUKSRLLF-QNRYFBKSSA-N 0.000 claims description 3
- FVMMQJUBNMOPPR-WLDMJGECSA-N N-[(3R,4R,5R,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)oxan-3-yl]formamide Chemical compound OC[C@H]1OC(O)[C@H](NC=O)[C@@H](O)[C@H]1O FVMMQJUBNMOPPR-WLDMJGECSA-N 0.000 claims description 3
- RTEOJYOKWPEKKN-HXQZNRNWSA-N N-[(3R,4R,5R,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)oxan-3-yl]propanamide Chemical compound CCC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O RTEOJYOKWPEKKN-HXQZNRNWSA-N 0.000 claims description 3
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 125000000837 carbohydrate group Chemical group 0.000 claims 1
- 108020004999 messenger RNA Proteins 0.000 abstract description 64
- 238000011282 treatment Methods 0.000 abstract description 62
- 108020000948 Antisense Oligonucleotides Proteins 0.000 abstract description 23
- 108020004414 DNA Proteins 0.000 description 136
- 150000001875 compounds Chemical class 0.000 description 89
- 125000005647 linker group Chemical group 0.000 description 84
- 241000699670 Mus sp. Species 0.000 description 60
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 55
- 101710163270 Nuclease Proteins 0.000 description 53
- 238000013461 design Methods 0.000 description 53
- 108010041986 DNA Vaccines Proteins 0.000 description 45
- 229940021995 DNA vaccine Drugs 0.000 description 45
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 45
- 210000001744 T-lymphocyte Anatomy 0.000 description 44
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 41
- 239000002953 phosphate buffered saline Substances 0.000 description 41
- 239000011780 sodium chloride Substances 0.000 description 41
- 229910052739 hydrogen Inorganic materials 0.000 description 39
- 239000001257 hydrogen Substances 0.000 description 39
- -1 at least 60% Chemical compound 0.000 description 35
- 150000001720 carbohydrates Chemical class 0.000 description 28
- 239000000203 mixture Substances 0.000 description 28
- 210000002966 serum Anatomy 0.000 description 28
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 24
- 238000003776 cleavage reaction Methods 0.000 description 24
- 230000007017 scission Effects 0.000 description 24
- 230000000694 effects Effects 0.000 description 23
- 241000282414 Homo sapiens Species 0.000 description 21
- 102100034349 Integrase Human genes 0.000 description 20
- 210000001519 tissue Anatomy 0.000 description 20
- 210000003494 hepatocyte Anatomy 0.000 description 19
- 241000699666 Mus <mouse, genus> Species 0.000 description 18
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 18
- 241001465754 Metazoa Species 0.000 description 17
- 230000005764 inhibitory process Effects 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 description 16
- 101710203526 Integrase Proteins 0.000 description 16
- 238000003556 assay Methods 0.000 description 16
- 235000014633 carbohydrates Nutrition 0.000 description 16
- 238000011529 RT qPCR Methods 0.000 description 15
- 239000002609 medium Substances 0.000 description 15
- 230000004048 modification Effects 0.000 description 15
- 238000012986 modification Methods 0.000 description 15
- 210000000822 natural killer cell Anatomy 0.000 description 15
- 229940115272 polyinosinic:polycytidylic acid Drugs 0.000 description 15
- 239000000523 sample Substances 0.000 description 15
- 230000001684 chronic effect Effects 0.000 description 14
- 239000003814 drug Substances 0.000 description 14
- 230000002708 enhancing effect Effects 0.000 description 14
- 230000001965 increasing effect Effects 0.000 description 14
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 13
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 13
- 241000700159 Rattus Species 0.000 description 13
- 244000052769 pathogen Species 0.000 description 13
- 238000011160 research Methods 0.000 description 13
- 125000006850 spacer group Chemical group 0.000 description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 239000003795 chemical substances by application Substances 0.000 description 12
- 210000004738 parenchymal cell Anatomy 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 108020004459 Small interfering RNA Proteins 0.000 description 11
- 230000005867 T cell response Effects 0.000 description 11
- 125000004103 aminoalkyl group Chemical group 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 11
- 239000000872 buffer Substances 0.000 description 11
- 239000002299 complementary DNA Substances 0.000 description 11
- 102000048776 human CD274 Human genes 0.000 description 11
- 239000000463 material Substances 0.000 description 11
- 230000001717 pathogenic effect Effects 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 11
- 102000004127 Cytokines Human genes 0.000 description 10
- 108090000695 Cytokines Proteins 0.000 description 10
- 208000035475 disorder Diseases 0.000 description 10
- 238000009396 hybridization Methods 0.000 description 10
- 150000002431 hydrogen Chemical class 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- 101001074035 Homo sapiens Zinc finger protein GLI2 Proteins 0.000 description 9
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 9
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 9
- 102100035558 Zinc finger protein GLI2 Human genes 0.000 description 9
- 230000021615 conjugation Effects 0.000 description 9
- 201000006747 infectious mononucleosis Diseases 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 210000000952 spleen Anatomy 0.000 description 9
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 8
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 8
- 208000036142 Viral infection Diseases 0.000 description 8
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 8
- 230000000692 anti-sense effect Effects 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical group NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 8
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 8
- 238000003197 gene knockdown Methods 0.000 description 8
- 230000036039 immunity Effects 0.000 description 8
- 230000002401 inhibitory effect Effects 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 8
- 238000007920 subcutaneous administration Methods 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 7
- 101710132601 Capsid protein Proteins 0.000 description 7
- 239000012981 Hank's balanced salt solution Substances 0.000 description 7
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 7
- 101001117316 Mus musculus Programmed cell death 1 ligand 1 Proteins 0.000 description 7
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 7
- 210000000612 antigen-presenting cell Anatomy 0.000 description 7
- 210000003719 b-lymphocyte Anatomy 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 230000036755 cellular response Effects 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000009826 distribution Methods 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 210000005229 liver cell Anatomy 0.000 description 7
- 230000001404 mediated effect Effects 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 210000005087 mononuclear cell Anatomy 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 230000003248 secreting effect Effects 0.000 description 7
- 239000002924 silencing RNA Substances 0.000 description 7
- 230000000638 stimulation Effects 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 230000009385 viral infection Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 241000282693 Cercopithecidae Species 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 6
- 239000004472 Lysine Substances 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 229920001213 Polysorbate 20 Polymers 0.000 description 6
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 6
- 102000003970 Vinculin Human genes 0.000 description 6
- 108090000384 Vinculin Proteins 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 239000012894 fetal calf serum Substances 0.000 description 6
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 6
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 6
- 238000004007 reversed phase HPLC Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- RHCSKNNOAZULRK-APZFVMQVSA-N 2,2-dideuterio-2-(3,4,5-trimethoxyphenyl)ethanamine Chemical compound NCC([2H])([2H])C1=CC(OC)=C(OC)C(OC)=C1 RHCSKNNOAZULRK-APZFVMQVSA-N 0.000 description 5
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 5
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 5
- 102100031780 Endonuclease Human genes 0.000 description 5
- 108010042407 Endonucleases Proteins 0.000 description 5
- 108010074328 Interferon-gamma Proteins 0.000 description 5
- 229930182816 L-glutamine Natural products 0.000 description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 5
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 229940104302 cytosine Drugs 0.000 description 5
- 230000007423 decrease Effects 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000012678 infectious agent Substances 0.000 description 5
- 210000003734 kidney Anatomy 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 238000002844 melting Methods 0.000 description 5
- 230000008018 melting Effects 0.000 description 5
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 5
- 210000003205 muscle Anatomy 0.000 description 5
- 210000000056 organ Anatomy 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
- 230000002265 prevention Effects 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 230000007115 recruitment Effects 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 125000001424 substituent group Chemical group 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 4
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 4
- 229930024421 Adenine Natural products 0.000 description 4
- 102000025850 HLA-A2 Antigen Human genes 0.000 description 4
- 108010074032 HLA-A2 Antigen Proteins 0.000 description 4
- 102100037850 Interferon gamma Human genes 0.000 description 4
- 108091027974 Mature messenger RNA Proteins 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 239000004698 Polyethylene Substances 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 239000003443 antiviral agent Substances 0.000 description 4
- 125000003118 aryl group Chemical group 0.000 description 4
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 125000000524 functional group Chemical group 0.000 description 4
- 125000001072 heteroaryl group Chemical group 0.000 description 4
- 238000007912 intraperitoneal administration Methods 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 238000002515 oligonucleotide synthesis Methods 0.000 description 4
- 150000008300 phosphoramidites Chemical class 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- 238000012552 review Methods 0.000 description 4
- 239000003161 ribonuclease inhibitor Substances 0.000 description 4
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 229940035893 uracil Drugs 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- 0 CC(C[*-]C1OC1CCOCC(COCCC(NCC(C)C[C@](C)CO[C@@](C(*)*1*)OC(CO)[C@@]1O)=O)(COCCC(N(C)C*(C)C*CO[C@@]1OC(CO)[C@](*)C([*+])C1*)=O)N*)CC(C)CO[C@@](C(*)C1O)OC(CO)[C@@]1O Chemical compound CC(C[*-]C1OC1CCOCC(COCCC(NCC(C)C[C@](C)CO[C@@](C(*)*1*)OC(CO)[C@@]1O)=O)(COCCC(N(C)C*(C)C*CO[C@@]1OC(CO)[C@](*)C([*+])C1*)=O)N*)CC(C)CO[C@@](C(*)C1O)OC(CO)[C@@]1O 0.000 description 3
- 101100463133 Caenorhabditis elegans pdl-1 gene Proteins 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 3
- 241000702421 Dependoparvovirus Species 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 108700024394 Exon Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 108091093037 Peptide nucleic acid Proteins 0.000 description 3
- 241000224016 Plasmodium Species 0.000 description 3
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000033289 adaptive immune response Effects 0.000 description 3
- 229960000643 adenine Drugs 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 125000004429 atom Chemical group 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 230000004700 cellular uptake Effects 0.000 description 3
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 230000003828 downregulation Effects 0.000 description 3
- 238000009509 drug development Methods 0.000 description 3
- 210000001163 endosome Anatomy 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010195 expression analysis Methods 0.000 description 3
- 150000002256 galaktoses Chemical class 0.000 description 3
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 210000001865 kupffer cell Anatomy 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 102000054765 polymorphisms of proteins Human genes 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000000651 prodrug Substances 0.000 description 3
- 229940002612 prodrug Drugs 0.000 description 3
- 238000011321 prophylaxis Methods 0.000 description 3
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 239000007929 subcutaneous injection Substances 0.000 description 3
- 238000010254 subcutaneous injection Methods 0.000 description 3
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 3
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- PMJWDPGOWBRILU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[4-(2,5-dioxopyrrol-1-yl)phenyl]butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCC(C=C1)=CC=C1N1C(=O)C=CC1=O PMJWDPGOWBRILU-UHFFFAOYSA-N 0.000 description 2
- WCMOHMXWOOBVMZ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCNC(=O)CCN1C(=O)C=CC1=O WCMOHMXWOOBVMZ-UHFFFAOYSA-N 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 2
- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 description 2
- 125000004454 (C1-C6) alkoxycarbonyl group Chemical group 0.000 description 2
- 125000004916 (C1-C6) alkylcarbonyl group Chemical group 0.000 description 2
- 125000006700 (C1-C6) alkylthio group Chemical group 0.000 description 2
- 125000006619 (C1-C6) dialkylamino group Chemical group 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- XGDRLCRGKUCBQL-UHFFFAOYSA-N 1h-imidazole-4,5-dicarbonitrile Chemical compound N#CC=1N=CNC=1C#N XGDRLCRGKUCBQL-UHFFFAOYSA-N 0.000 description 2
- RUVRGYVESPRHSZ-UHFFFAOYSA-N 2-[2-(2-azaniumylethoxy)ethoxy]acetate Chemical compound NCCOCCOCC(O)=O RUVRGYVESPRHSZ-UHFFFAOYSA-N 0.000 description 2
- QUBNFZFTFXTLKH-UHFFFAOYSA-N 2-aminododecanoic acid Chemical compound CCCCCCCCCCC(N)C(O)=O QUBNFZFTFXTLKH-UHFFFAOYSA-N 0.000 description 2
- 108020005065 3' Flanking Region Proteins 0.000 description 2
- DRNGLYHKYPNTEA-UHFFFAOYSA-N 4-azaniumylcyclohexane-1-carboxylate Chemical compound NC1CCC(C(O)=O)CC1 DRNGLYHKYPNTEA-UHFFFAOYSA-N 0.000 description 2
- 108020005029 5' Flanking Region Proteins 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 108091029792 Alkylated DNA Proteins 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- 101150075175 Asgr1 gene Proteins 0.000 description 2
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 2
- 125000000882 C2-C6 alkenyl group Chemical group 0.000 description 2
- 125000003320 C2-C6 alkenyloxy group Chemical group 0.000 description 2
- 125000003601 C2-C6 alkynyl group Chemical group 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 102000000844 Cell Surface Receptors Human genes 0.000 description 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- NBSCHQHZLSJFNQ-QTVWNMPRSA-N D-Mannose-6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@@H]1O NBSCHQHZLSJFNQ-QTVWNMPRSA-N 0.000 description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 2
- 238000011238 DNA vaccination Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000005977 Ethylene Substances 0.000 description 2
- 101000785944 Homo sapiens Asialoglycoprotein receptor 1 Proteins 0.000 description 2
- 101000917826 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-a Proteins 0.000 description 2
- 101000917824 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-b Proteins 0.000 description 2
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 2
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 2
- 101001023379 Homo sapiens Lysosome-associated membrane glycoprotein 1 Proteins 0.000 description 2
- 101000693243 Homo sapiens Paternally-expressed gene 3 protein Proteins 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- ZGUNAGUHMKGQNY-ZETCQYMHSA-N L-alpha-phenylglycine zwitterion Chemical compound OC(=O)[C@@H](N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-ZETCQYMHSA-N 0.000 description 2
- 241000222722 Leishmania <genus> Species 0.000 description 2
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 2
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 2
- 102100035133 Lysosome-associated membrane glycoprotein 1 Human genes 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 102100025757 Paternally-expressed gene 3 protein Human genes 0.000 description 2
- 208000037581 Persistent Infection Diseases 0.000 description 2
- 241000223960 Plasmodium falciparum Species 0.000 description 2
- 241000223821 Plasmodium malariae Species 0.000 description 2
- 206010035501 Plasmodium malariae infection Diseases 0.000 description 2
- 241000223810 Plasmodium vivax Species 0.000 description 2
- 108091036414 Polyinosinic:polycytidylic acid Proteins 0.000 description 2
- 101710094000 Programmed cell death 1 ligand 1 Proteins 0.000 description 2
- 238000011530 RNeasy Mini Kit Methods 0.000 description 2
- 239000012979 RPMI medium Substances 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 101710137302 Surface antigen S Proteins 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 241000223997 Toxoplasma gondii Species 0.000 description 2
- 241000223105 Trypanosoma brucei Species 0.000 description 2
- 241000223109 Trypanosoma cruzi Species 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 206010058874 Viraemia Diseases 0.000 description 2
- 108010067390 Viral Proteins Proteins 0.000 description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- WOZSCQDILHKSGG-UHFFFAOYSA-N adefovir depivoxil Chemical compound N1=CN=C2N(CCOCP(=O)(OCOC(=O)C(C)(C)C)OCOC(=O)C(C)(C)C)C=NC2=C1N WOZSCQDILHKSGG-UHFFFAOYSA-N 0.000 description 2
- 229960005305 adenosine Drugs 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 125000005129 aryl carbonyl group Chemical group 0.000 description 2
- 125000005161 aryl oxy carbonyl group Chemical group 0.000 description 2
- 125000004104 aryloxy group Chemical group 0.000 description 2
- 229960003852 atezolizumab Drugs 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 229940000635 beta-alanine Drugs 0.000 description 2
- 125000002619 bicyclic group Chemical group 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- KQNZDYYTLMIZCT-KQPMLPITSA-N brefeldin A Chemical compound O[C@@H]1\C=C\C(=O)O[C@@H](C)CCC\C=C\[C@@H]2C[C@H](O)C[C@H]21 KQNZDYYTLMIZCT-KQPMLPITSA-N 0.000 description 2
- JUMGSHROWPPKFX-UHFFFAOYSA-N brefeldin-A Natural products CC1CCCC=CC2(C)CC(O)CC2(C)C(O)C=CC(=O)O1 JUMGSHROWPPKFX-UHFFFAOYSA-N 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 210000000234 capsid Anatomy 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 108091092328 cellular RNA Proteins 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 125000003636 chemical group Chemical group 0.000 description 2
- 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 2
- 229960002424 collagenase Drugs 0.000 description 2
- 238000002648 combination therapy Methods 0.000 description 2
- 239000013068 control sample Substances 0.000 description 2
- 230000001461 cytolytic effect Effects 0.000 description 2
- 238000004163 cytometry Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 229940088679 drug related substance Drugs 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- QDGZDCVAUDNJFG-FXQIFTODSA-N entecavir (anhydrous) Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@H]1C[C@H](O)[C@@H](CO)C1=C QDGZDCVAUDNJFG-FXQIFTODSA-N 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 2
- 230000009368 gene silencing by RNA Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 229910052736 halogen Inorganic materials 0.000 description 2
- 150000002367 halogens Chemical class 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 125000005223 heteroarylcarbonyl group Chemical group 0.000 description 2
- 125000005553 heteroaryloxy group Chemical group 0.000 description 2
- 125000005226 heteroaryloxycarbonyl group Chemical group 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 125000001165 hydrophobic group Chemical group 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000012606 in vitro cell culture Methods 0.000 description 2
- 230000015788 innate immune response Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 238000007913 intrathecal administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000000111 isothermal titration calorimetry Methods 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 230000002934 lysing effect Effects 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 210000003712 lysosome Anatomy 0.000 description 2
- 230000001868 lysosomic effect Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 2
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 210000001087 myotubule Anatomy 0.000 description 2
- 210000000581 natural killer T-cell Anatomy 0.000 description 2
- 238000007899 nucleic acid hybridization Methods 0.000 description 2
- 230000000269 nucleophilic effect Effects 0.000 description 2
- PBLZLIFKVPJDCO-UHFFFAOYSA-N omega-Aminododecanoic acid Natural products NCCCCCCCCCCCC(O)=O PBLZLIFKVPJDCO-UHFFFAOYSA-N 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000010412 perfusion Effects 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
- 229920000642 polymer Polymers 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- 238000000751 protein extraction Methods 0.000 description 2
- 150000003212 purines Chemical class 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000012772 sequence design Methods 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 108010062513 snake venom phosphodiesterase I Proteins 0.000 description 2
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 description 2
- 229910001488 sodium perchlorate Inorganic materials 0.000 description 2
- 229940054269 sodium pyruvate Drugs 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- IQFYYKKMVGJFEH-CSMHCCOUSA-N telbivudine Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1O[C@@H](CO)[C@H](O)C1 IQFYYKKMVGJFEH-CSMHCCOUSA-N 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- TYKASZBHFXBROF-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-(2,5-dioxopyrrol-1-yl)acetate Chemical compound O=C1CCC(=O)N1OC(=O)CN1C(=O)C=CC1=O TYKASZBHFXBROF-UHFFFAOYSA-N 0.000 description 1
- VLARLSIGSPVYHX-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-(2,5-dioxopyrrol-1-yl)hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCN1C(=O)C=CC1=O VLARLSIGSPVYHX-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
- GUAHPAJOXVYFON-ZETCQYMHSA-N (8S)-8-amino-7-oxononanoic acid zwitterion Chemical compound C[C@H](N)C(=O)CCCCCC(O)=O GUAHPAJOXVYFON-ZETCQYMHSA-N 0.000 description 1
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 1
- HBOMLICNUCNMMY-KJFJCRTCSA-N 1-[(4s,5s)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-KJFJCRTCSA-N 0.000 description 1
- 102100040685 14-3-3 protein zeta/delta Human genes 0.000 description 1
- XQCZBXHVTFVIFE-UHFFFAOYSA-N 2-amino-4-hydroxypyrimidine Chemical compound NC1=NC=CC(O)=N1 XQCZBXHVTFVIFE-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
- HBJGQJWNMZDFKL-UHFFFAOYSA-N 2-chloro-7h-purin-6-amine Chemical compound NC1=NC(Cl)=NC2=C1NC=N2 HBJGQJWNMZDFKL-UHFFFAOYSA-N 0.000 description 1
- KMEMIMRPZGDOMG-UHFFFAOYSA-N 2-cyanoethoxyphosphonamidous acid Chemical class NP(O)OCCC#N KMEMIMRPZGDOMG-UHFFFAOYSA-N 0.000 description 1
- YBANXOPIYSVPMH-UHFFFAOYSA-N 3-[[di(propan-2-yl)amino]-[6-[[(4-methoxyphenyl)-diphenylmethyl]amino]hexoxy]phosphanyl]oxypropanenitrile Chemical compound C1=CC(OC)=CC=C1C(NCCCCCCOP(OCCC#N)N(C(C)C)C(C)C)(C=1C=CC=CC=1)C1=CC=CC=C1 YBANXOPIYSVPMH-UHFFFAOYSA-N 0.000 description 1
- 125000002103 4,4'-dimethoxytriphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)(C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H])C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H] 0.000 description 1
- FLCPHISGULLHCH-UHFFFAOYSA-N 4-amino-2-(6-aminohexoxy)butanoic acid Chemical compound NCCCCCCOC(C(=O)O)CCN FLCPHISGULLHCH-UHFFFAOYSA-N 0.000 description 1
- JYCQQPHGFMYQCF-UHFFFAOYSA-N 4-tert-Octylphenol monoethoxylate Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCO)C=C1 JYCQQPHGFMYQCF-UHFFFAOYSA-N 0.000 description 1
- YWZHEXZIISFIDA-UHFFFAOYSA-N 5-amino-1,2,4-dithiazole-3-thione Chemical compound NC1=NC(=S)SS1 YWZHEXZIISFIDA-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
- 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
- UJBCLAXPPIDQEE-UHFFFAOYSA-N 5-prop-1-ynyl-1h-pyrimidine-2,4-dione Chemical compound CC#CC1=CNC(=O)NC1=O UJBCLAXPPIDQEE-UHFFFAOYSA-N 0.000 description 1
- QSHQKIURKJITMZ-OBUPQJQESA-N 5β-cholane Chemical compound C([C@H]1CC2)CCC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)CCC)[C@@]2(C)CC1 QSHQKIURKJITMZ-OBUPQJQESA-N 0.000 description 1
- QNNARSZPGNJZIX-UHFFFAOYSA-N 6-amino-5-prop-1-ynyl-1h-pyrimidin-2-one Chemical compound CC#CC1=CNC(=O)N=C1N QNNARSZPGNJZIX-UHFFFAOYSA-N 0.000 description 1
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- VKKXEIQIGGPMHT-UHFFFAOYSA-N 7h-purine-2,8-diamine Chemical compound NC1=NC=C2NC(N)=NC2=N1 VKKXEIQIGGPMHT-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
- 241000702423 Adeno-associated virus - 2 Species 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 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
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 101100421761 Arabidopsis thaliana GSNAP gene Proteins 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- RAFGOPNHDSXPQA-YAIWYBQCSA-N B[C@@H]1O[C@@]2(COC)CN(C)[C@H]1C2OC.B[C@@H]1O[C@@]2(COC)CN[C@H]1C2OC.B[C@@H]1O[C@@]2(COC)CO[C@H]1C2OC.B[C@@H]1O[C@@]2(COC)CS[C@H]1C2OC.B[C@@H]1O[C@]2(COC)CN[C@@H]1C2OC.B[C@@H]1O[C@]2(COC)CO[C@@H]1C2OC.B[C@@H]1O[C@]2(COC)CS[C@@H]1C2OC Chemical compound B[C@@H]1O[C@@]2(COC)CN(C)[C@H]1C2OC.B[C@@H]1O[C@@]2(COC)CN[C@H]1C2OC.B[C@@H]1O[C@@]2(COC)CO[C@H]1C2OC.B[C@@H]1O[C@@]2(COC)CS[C@H]1C2OC.B[C@@H]1O[C@]2(COC)CN[C@@H]1C2OC.B[C@@H]1O[C@]2(COC)CO[C@@H]1C2OC.B[C@@H]1O[C@]2(COC)CS[C@@H]1C2OC RAFGOPNHDSXPQA-YAIWYBQCSA-N 0.000 description 1
- 231100000699 Bacterial toxin Toxicity 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 102100026031 Beta-glucuronidase Human genes 0.000 description 1
- 101000588922 Bunodosoma cangicum Delta-actitoxin-Bcg1a Proteins 0.000 description 1
- CRZFCSKIHRANCM-UUIBEKTBSA-N C=CCO[C@H]1C(OC)[C@@H](COC)O[C@H]1C.COCCO[C@H]1C(OC)[C@@H](COC)O[C@H]1C.COC[C@H]1O[C@@H](C)[C@@H](F)C1OC.COC[C@H]1O[C@@H](C)[C@@H](OC)C1OC.COC[C@H]1O[C@@H](C)[C@@H](OCCN)C1OC.COC[C@H]1O[C@@H](C)[C@H](F)C1OC Chemical compound C=CCO[C@H]1C(OC)[C@@H](COC)O[C@H]1C.COCCO[C@H]1C(OC)[C@@H](COC)O[C@H]1C.COC[C@H]1O[C@@H](C)[C@@H](F)C1OC.COC[C@H]1O[C@@H](C)[C@@H](OC)C1OC.COC[C@H]1O[C@@H](C)[C@@H](OCCN)C1OC.COC[C@H]1O[C@@H](C)[C@H](F)C1OC CRZFCSKIHRANCM-UUIBEKTBSA-N 0.000 description 1
- 210000004366 CD4-positive T-lymphocyte Anatomy 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102100021868 Calnexin Human genes 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241001432959 Chernes Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108020004394 Complementary RNA Proteins 0.000 description 1
- 229940126062 Compound A Drugs 0.000 description 1
- 108010062580 Concanavalin A Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 101150018489 DNA-C gene Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 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 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102100030013 Endoribonuclease Human genes 0.000 description 1
- 108010093099 Endoribonucleases Proteins 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical group C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 206010015548 Euthanasia Diseases 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 101001114134 Gloydius halys Neutral phospholipase A2 agkistrodotoxin Proteins 0.000 description 1
- 101150001754 Gusb gene Proteins 0.000 description 1
- 241000285366 HBV genotype D Species 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- NLDMNSXOCDLTTB-UHFFFAOYSA-N Heterophylliin A Natural products O1C2COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)C(O)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 NLDMNSXOCDLTTB-UHFFFAOYSA-N 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 101000964898 Homo sapiens 14-3-3 protein zeta/delta Proteins 0.000 description 1
- 101100215371 Homo sapiens ACTB gene Proteins 0.000 description 1
- 101000785948 Homo sapiens Asialoglycoprotein receptor 2 Proteins 0.000 description 1
- 101000933465 Homo sapiens Beta-glucuronidase Proteins 0.000 description 1
- 101000898052 Homo sapiens Calnexin Proteins 0.000 description 1
- 101000742236 Homo sapiens Vitamin K-dependent gamma-carboxylase Proteins 0.000 description 1
- 241000700588 Human alphaherpesvirus 1 Species 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
- 108700002232 Immediate-Early Genes Proteins 0.000 description 1
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 1
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-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
- 101710084021 Large envelope protein Proteins 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 206010024305 Leukaemia monocytic Diseases 0.000 description 1
- 241000712899 Lymphocytic choriomeningitis mammarenavirus Species 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 241000710118 Maize chlorotic mottle virus Species 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 229930191564 Monensin Natural products 0.000 description 1
- GAOZTHIDHYLHMS-UHFFFAOYSA-N Monensin A Natural products O1C(CC)(C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)CCC1C(O1)(C)CCC21CC(O)C(C)C(C(C)C(OC)C(C)C(O)=O)O2 GAOZTHIDHYLHMS-UHFFFAOYSA-N 0.000 description 1
- 229910003827 NRaRb Inorganic materials 0.000 description 1
- 101000783356 Naja sputatrix Cytotoxin Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 241000360071 Pituophis catenifer Species 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010035742 Pneumonitis Diseases 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 230000037453 T cell priming Effects 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical compound OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000223104 Trypanosoma Species 0.000 description 1
- 102100038182 Vitamin K-dependent gamma-carboxylase Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000002730 additional effect Effects 0.000 description 1
- 229960001997 adefovir Drugs 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229960002684 aminocaproic acid Drugs 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000009175 antibody therapy Methods 0.000 description 1
- 239000008365 aqueous carrier Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 239000000688 bacterial toxin Substances 0.000 description 1
- 229940002637 baraclude 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
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000002340 cardiotoxin Substances 0.000 description 1
- 231100000677 cardiotoxin Toxicity 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 150000002009 diols Chemical class 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 229960000980 entecavir Drugs 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 210000004024 hepatic stellate cell Anatomy 0.000 description 1
- 230000003494 hepatotrophic effect Effects 0.000 description 1
- 229940097709 hepsera Drugs 0.000 description 1
- 150000002402 hexoses Chemical group 0.000 description 1
- 239000011539 homogenization buffer Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000001841 imino group Chemical group [H]N=* 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 210000005007 innate immune system Anatomy 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000010212 intracellular staining Methods 0.000 description 1
- 238000000185 intracerebroventricular administration Methods 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- JTEGQNOMFQHVDC-NKWVEPMBSA-N lamivudine Chemical compound O=C1N=C(N)C=CN1[C@H]1O[C@@H](CO)SC1 JTEGQNOMFQHVDC-NKWVEPMBSA-N 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 229960005358 monensin Drugs 0.000 description 1
- GAOZTHIDHYLHMS-KEOBGNEYSA-N monensin A Chemical compound C([C@@](O1)(C)[C@H]2CC[C@@](O2)(CC)[C@H]2[C@H](C[C@@H](O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C[C@@]21C[C@H](O)[C@@H](C)[C@@H]([C@@H](C)[C@@H](OC)[C@H](C)C(O)=O)O2 GAOZTHIDHYLHMS-KEOBGNEYSA-N 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 229940127073 nucleoside analogue Drugs 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 125000001117 oleyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 125000004043 oxo group Chemical group O=* 0.000 description 1
- 125000001312 palmitoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229940002988 pegasys Drugs 0.000 description 1
- 108010092853 peginterferon alfa-2a Proteins 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 1
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical compound OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 description 1
- 125000005642 phosphothioate group Chemical group 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 210000003240 portal vein Anatomy 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 description 1
- 244000000040 protozoan parasite Species 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 238000001303 quality assessment method Methods 0.000 description 1
- 238000013442 quality metrics Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 229960000329 ribavirin Drugs 0.000 description 1
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 238000011451 sequencing strategy Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000012421 spiking Methods 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 210000004500 stellate cell Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 229960005311 telbivudine Drugs 0.000 description 1
- SGOIRFVFHAKUTI-ZCFIWIBFSA-N tenofovir (anhydrous) Chemical compound N1=CN=C2N(C[C@@H](C)OCP(O)(O)=O)C=NC2=C1N SGOIRFVFHAKUTI-ZCFIWIBFSA-N 0.000 description 1
- 229940021747 therapeutic vaccine Drugs 0.000 description 1
- 238000006177 thiolation reaction Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- ZEMGGZBWXRYJHK-UHFFFAOYSA-N thiouracil Chemical compound O=C1C=CNC(=S)N1 ZEMGGZBWXRYJHK-UHFFFAOYSA-N 0.000 description 1
- 125000000464 thioxo group Chemical group S=* 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 229940044616 toll-like receptor 7 agonist Drugs 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000006433 tumor necrosis factor production Effects 0.000 description 1
- 229940063032 tyzeka Drugs 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 1
- 229960001600 xylazine Drugs 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1131—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 against viruses
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
-
- 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
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- 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
-
- 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
-
- 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
-
- 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
- A61P31/20—Antivirals for DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
- A61P33/06—Antimalarials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
-
- 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
-
- 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
- C12N15/1138—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 against receptors or cell surface proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/322—2'-R Modification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/323—Chemical structure of the sugar modified ring structure
- C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/34—Spatial arrangement of the modifications
- C12N2310/341—Gapmers, i.e. of the type ===---===
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/34—Spatial arrangement of the modifications
- C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/35—Nature of the modification
- C12N2310/351—Conjugate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to oligonucleotides (oligomers) that are complementary to programmed death ligand-1 (PD-L1), leading to reduction of the expression of PD-L1 the liver.
- the present invention also relates to a method of alleviating the T cell exhaustion caused by an infection of the liver or cancer in the liver.
- Relevant infections are chronic HBV, HCV and HDV and parasite infections like malaria and toxoplasmosis (e.g. caused by protozoa of the Plasmodium , in particular of the species P. vivax, P. malariae and P. falciparum ).
- the costimulatory pathway consisting of the programmed death-1 (PD-1) receptor and its ligand, PD-L1 (or B7-H1 or CD274) is known to contribute directly to T cell exhaustion resulting in lack of viral control during chronic infections of the liver.
- the PD-1 pathway also plays a role in autoimmunity as mice disrupted in this pathway develop autoimmune diseases.
- WO 2006/042237 describes a method of diagnosing cancer by assessing PD-L1 (B7-H1) expression in tumors and suggests delivering an agent, which interferes with the PD-1/PD-L1 interaction, to a patient.
- Interfering agents can be antibodies, antibody fragments, siRNA or antisense oligonucleotides. There are no specific examples of such interfering agents nor is there any mentioning of chronic liver infections.
- RNA interference mediated inhibition of PD-L1 using double stranded RNA (dsRNA, RNAi or siRNA) molecules have also been disclosed in for example WO 2005/007855, WO 2007/084865 and U.S. Pat. No. 8,507,663. None of these describes targeted delivery to the liver.
- siRNA approach is significantly different from the single stranded antisense oligonucleotide approach since the biodistribution and the mode of actions is quite different.
- antisense oligonucleotides and siRNAs have different preferences for target sites in the mRNA.
- WO2016/138278 describes inhibition of immune checkpoints including PD-L1, using two or more single stranded antisense oligonucleotides that are linked at their 5′ ends.
- the application does not mention HBV or targeted delivery to the liver.
- the present invention identifies novel oligonucleotides and oligonucleotide conjugates which reduce PD-L1 mRNA very efficiently in liver cells, both in parenchymal cells (e.g. hepatocytes) and in non-parenchymal cells such as Kupffer cells and liver sinusoidal endothelial cells (LSECs).
- parenchymal cells e.g. hepatocytes
- non-parenchymal cells e.g. Kupffer cells and liver sinusoidal endothelial cells (LSECs).
- LSECs liver sinusoidal endothelial cells
- Natural killer (NK) cells and natural killer T (NKT) cells may also be activated by the oligonucleotides and oligonucleotide conjugates of the present invention.
- the oligonucleotide conjugates secures local reduction of PD-L1 in liver cells and therefore reduces the risk of autoimmune side effects, such as pneumonitis, non-viral hepatitis and colitis associated with systemic depletion of PD-L1.
- the present invention relates to oligonucleotides or conjugates thereof targeting a nucleic acid capable of modulating the expression of PD-L1 and to treat or prevent diseases related to the functioning of the PD-L1.
- the oligonucleotides or oligonucleotide conjugates may in particular be used to treat diseases where the immune response against an infectious agent has been exhausted.
- the invention provides oligonucleotides which comprise a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementarity to a PD-L1 target nucleic acid.
- the oligonucleotide can be an antisense oligonucleotide, preferably with a gapmer design.
- the oligonucleotide is capable of inhibiting the expression of PD-L1 by cleavage of a target nucleic acid. The cleavage is preferably achieved via nuclease recruitment.
- the oligonucleotide is conjugated to at least one asialoglycoprotein receptor targeting conjugate moiety, such as a conjugate moiety comprising at least one N-Acetylgalactosamine (GalNAc) moiety.
- the conjugation moiety and the oligonucleotide may be linked together by a linker, in particular a biocleavable linker.
- the invention provides pharmaceutical compositions comprising the oligonucleotides or oligonucleotide conjugates of the invention and pharmaceutically acceptable diluents, carriers, salts and/or adjuvants.
- the invention provides methods for in vivo or in vitro method for reduction of PD-L1 expression in a target cell which is expressing PD-L1, by administering an oligonucleotide or composition of the invention in an effective amount to said cell.
- the invention provides oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for use in restoration of immunity against a virus or parasite.
- the invention provides oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for use as a medicament.
- the invention provides methods for treating or preventing a disease, disorder or dysfunction by administering a therapeutically or prophylactically effective amount of the oligonucleotide of the invention to a subject suffering from or susceptible to the disease, disorder or dysfunction, in particular diseases selected from viral liver infections or parasite infections.
- oligonucleotide, oligonucleotide conjugates or pharmaceutical composition of the invention is used in the treatment or prevention of viral liver infections such as HBV, HCV and HDV or a parasite infections such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis or liver cancer or metastases in the liver.
- viral liver infections such as HBV, HCV and HDV
- a parasite infections such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis or liver cancer or metastases in the liver.
- FIG. 1 Illustrates exemplary antisense oligonucleotide conjugates, where the oligonucleotide either is represented as a wavy line (A-D) or as “oligonucleotide” (E-H) or as T 2 (I) and the asialoglycoprotein receptor targeting conjugate moieties are trivalent N-acetylgalactosamine moieties.
- Compounds A to D comprise a di-lysine brancher molecule a PEG3 spacer and three terminal GalNAc carbohydrate moieties.
- the oligonucleotide is attached directly to the asialoglycoprotein receptor targeting conjugate moiety without a linker.
- oligonucleotide is attached directly to the asialoglycoprotein receptor targeting conjugate moiety via a C6 linker.
- Compounds E-I comprise a trebler brancher molecule and spacers of varying length and structure and three terminal GalNAc carbohydrate moieties.
- FIG. 2 Graph showing EC50 (A) and PD-L1 knock down as % of saline (B) for the compounds tested in Example 2, in relation to their position on the target nucleic acid.
- the cell line in which the compound were tested are THP1( ⁇ ) and Karpas (*).
- FIG. 3 Structural formula of the trivalent GalNAc cluster (GN2).
- GN2 is useful as conjugation moiety in the present invention.
- the wavy line illustrates the site of conjugation of the cluster to e.g. a C6 amino linker or directly to the oligonucleotide.
- FIG. 4 Structural formula of CMP ID NO 766_2.
- FIG. 5 Structural formula of CMP ID NO 767_2.
- FIG. 6 Structural formula of CMP ID NO 768_2.
- FIG. 7 Structural formula of CMP ID NO 769_2.
- FIG. 8 Structural formula of CMP ID NO 770_2.
- FIG. 9 Western blot detecting PD-L1 protein expression in liver from poly(IC) induced animals following treatment with saline and the indicated CMP ID NO's.
- Each blot shows a naked oligonucleotide versus a GalNAc conjugated version of the same oligonucleotide, blot A) CMP ID NO 744_1 and 755_2, B) CMP ID NO 747_1 and 758_2, C) CMP ID NO 748_1 and 759_2, D) CMP ID NO 752_1 and 763_2 and E) CMP ID NO 753_1 and 764_2.
- the upper band is the vinculin loading control
- the lower band is the PD-L1 protein.
- the first lane in each blot show saline treated mice without Poly(IC) induction. These mice express very little PD-L protein.
- FIG. 10 Population of mononuclear cells in the liver after treatment with ⁇ vehicle (group 10 and 1), ⁇ DNA vaccine (group 11 and 2), ⁇ anti-PD-L1 antibody (group 12), ⁇ naked PD-L1 ASO+DNA vaccine (group 7) or ⁇ GalNAc conjugated PD-L1 ASO+DNA vaccine (group 8), for each group the individual animals are represented and the average is indicated by the vertical line for each group (see table 18).
- A) represents the number of T cells in the liver following treatment.
- B) represents the fraction of CD4+ T cells
- C) represents the fraction of CD8+ T cells.
- A) represents the percentage of CD8+ T cells which express PD-L1 in the liver following treatment.
- B) represents the percentage of CD4+ T cells which express PD-L1 in the liver following treatment
- C) represents the percentage of B cells which express PD-L1 in the liver following treatment.
- A) represents the percentage of IFN- ⁇ secreting CD8+ T cells in the liver which are specific towards HBV PreS2+S antigen following treatment.
- B) represents the percentage of IFN- ⁇ secreting CD8+ T cells in the liver which are specific towards HBV core antigen following treatment and
- C) represents the percentage of IFN- ⁇ and TNF- ⁇ secreting CD8+ T cells in the liver which are specific towards HBV PreS2+S antigen following treatment.
- FIG. 13 HBV-DNA, HBsAg and HBeAg in AAV/HBV mice following treatment with GalNAc conjugated PD-L1 antisense CMP NO: 759_2 ( ⁇ ) compared to vehicle ( ⁇ ).
- the vertical line indicates the end of treatment.
- oligonucleotide as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides.
- the oligonucleotide of the invention is man-made, and is chemically synthesized, and is typically purified or isolated.
- the oligonucleotide of the invention may comprise one or more modified nucleosides or nucleotides.
- Antisense oligonucleotide as used herein is defined as oligonucleotides capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid.
- the antisense oligonucleotides are not essentially double stranded and are therefore not siRNAs.
- the antisense oligonucleotides of the present invention are single stranded.
- oligonucleotide sequence refers to the region of the oligonucleotide which is complementary to the target nucleic acid.
- the term is used interchangeably herein with the term “contiguous nucleobase sequence” and the term “oligonucleotide motif sequence”.
- all the nucleotides of the oligonucleotide constitute the contiguous nucleotide sequence.
- the oligonucleotide comprises the contiguous nucleotide sequence and may optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used to attach a functional group to the contiguous nucleotide sequence.
- the nucleotide linker region may or may not be complementary to the target nucleic acid.
- Nucleotides are the building blocks of oligonucleotides and polynucleotides and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides.
- nucleotides such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides).
- Nucleosides and nucleotides may also interchangeably be referred to as “units” or “monomers”.
- modified nucleoside or “nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety.
- the modified nucleoside comprise a modified sugar moiety.
- modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue” or modified “units” or modified “monomers”.
- modified internucleoside linkage is defined as generally understood by the skilled person as linkages other than phosphodiester (PO) linkages, that covalently couples two nucleosides together. Nucleotides with modified internucleoside linkage are also termed “modified nucleotides”. In some embodiments, the modified internucleoside linkage increases the nuclease resistance of the oligonucleotide compared to a phosphodiester linkage. For naturally occurring oligonucleotides, the internucleoside linkage includes phosphate groups creating a phosphodiester bond between adjacent nucleosides.
- Modified internucleoside linkages are particularly useful in stabilizing oligonucleotides for in vivo use, and may serve to protect against nuclease cleavage at regions of DNA or RNA nucleosides in the oligonucleotide of the invention, for example within the gap region of a gapmer oligonucleotide, as well as in regions of modified nucleosides.
- the oligonucleotide comprises one or more internucleoside linkages modified from the natural phosphodiester to a linkage that is for example more resistant to nuclease attack.
- Nuclease resistance may be determined by incubating the oligonucleotide in blood serum or by using a nuclease resistance assay (e.g. snake venom phosphodiesterase (SVPD)), both are well known in the art.
- SVPD snake venom phosphodiesterase
- Internucleoside linkages which are capable of enhancing the nuclease resistance of an oligonucleotide are referred to as nuclease resistant internucleoside linkages.
- At least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof are modified, such as at least 60%, such as at least 70%, such as at least 80 or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are modified. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are modified.
- nucleosides which link the oligonucleotide of the invention to a non-nucleotide functional group, such as a conjugate may be phosphodiester.
- all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages.
- Modified internucleoside linkages may be selected from the group comprising phosphorothioate, diphosphorothioate and boranophosphate.
- the modified internucleoside linkages are compatible with the RNaseH recruitment of the oligonucleotide of the invention, for example phosphorothioate, diphosphorothioate or boranophosphate.
- the internucleoside linkage comprises sulphur (S), such as a phosphorothioate internucleoside linkage.
- a phosphorothioate internucleoside linkage is particularly useful due to nuclease resistance, beneficial pharmakokinetics and ease of manufacture.
- at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 80 or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate.
- all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof are phosphorothioate.
- the oligonucleotide comprises one or more neutral internucleoside linkage, particularly a internucleoside linkage selected from phosphotriester, methylphosphonate, MMI, amide-3, formacetal or thioformacetal.
- internucleoside linkages are disclosed in WO2009/124238 (incorporated herein by reference).
- the internucleoside linkage is selected from linkers disclosed in WO2007/031091 (incorporated herein by reference).
- the internucleoside linkage may be selected from —O—P(O) 2 —O—, —O—P(O,S)—O—, —O—P(S) 2 —O—, —S—P(O,S)—O—, —S—P(O,S)—O—, —S—P(O) 2 —O—, —O—P(O) 2 —S—, —O—P(O,S)—S—, —O—PO(R H )—O—, 0-PO(OCH 3 )—0-, —O—PO(NR H )—O—, —O—PO(OCH 2 CH 2 S—R)—O—
- Nuclease resistant linkages such as phosphothioate linkages, are particularly useful in oligonucleotide regions capable of recruiting nuclease when forming a duplex with the target nucleic acid, such as region G for gapmers, or the non-modified nucleoside region of headmers and tailmers.
- Phosphorothioate linkages may, however, also be useful in non-nuclease recruiting regions and/or affinity enhancing regions such as regions F and F′ for gapmers, or the modified nucleoside region of headmers and tailmers.
- Each of the design regions may however comprise internucleoside linkages other than phosphorothioate, such as phosphodiester linkages, in particularly in regions where modified nucleosides, such as LNA, protect the linkage against nuclease degradation.
- phosphodiester linkages such as one or two linkages, particularly between or adjacent to modified nucleoside units (typically in the non-nuclease recruiting regions) can modify the bioavailability and/or bio-distribution of an oligonucleotide—see WO2008/113832, incorporated herein by reference.
- all the internucleoside linkages in the oligonucleotide are phosphorothioate and/or boranophosphate linkages.
- all the internucleoside linkages in the oligonucleotide are phosphorothioate linkages.
- nucleobase includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization.
- pyrimidine e.g. uracil, thymine and cytosine
- nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization.
- nucleobase refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.
- the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobased selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2′thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.
- a nucleobased selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-brom
- the nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or U, wherein each letter may optionally include modified nucleobases of equivalent function.
- the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine.
- 5-methyl cytosine LNA nucleosides may be used.
- modified oligonucleotide describes an oligonucleotide comprising one or more sugar-modified nucleosides and/or modified internucleoside linkages.
- chimeric oligonucleotide is a term that has been used in the literature to describe oligonucleotides with modified nucleosides.
- Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T)/uracil (U).
- oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).
- % complementary refers to the number of nucleotides in percent of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which, at a given position, are complementary to (i.e. form Watson Crick base pairs with) a contiguous nucleotide sequence, at a given position of a separate nucleic acid molecule (e.g. the target nucleic acid).
- the percentage is calculated by counting the number of aligned bases that form pairs between the two sequences (when aligned with the target sequence 5′-3′ and the oligonucleotide sequence from 3′-5′), dividing by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such a comparison a nucleobase/nucleotide which does not align (form a base pair) is termed a mismatch.
- oligonucleotide SEQ ID NO: 5
- target nucleic acid SEQ ID NO: 772
- Identity refers to the number of nucleotides in percent of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which, at a given position, are identical to (i.e. in their ability to form Watson Crick base pairs with the complementary nucleoside) a contiguous nucleotide sequence, at a given position of a separate nucleic acid molecule (e.g. the target nucleic acid).
- the percentage is calculated by counting the number of aligned bases that are identical between the two sequences, including gaps, dividing by the total number of nucleotides in the oligonucleotide and multiplying by 100.
- Percent Identity (Matches ⁇ 100)/Length of aligned region (with gaps).
- hybridizing or “hybridizes” as used herein is to be understood as two nucleic acid strands (e.g. an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex.
- the affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (T m ) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions T m is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537).
- ⁇ G° is the energy associated with a reaction where aqueous concentrations are 1M, the pH is 7, and the temperature is 37° C.
- ⁇ G° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC) method as described in Hansen et al., 1965 , Chem. Comm. 36-38 and Holdgate et al., 2005 , Drug Discov Today. The skilled person will know that commercial equipment is available for ⁇ G° measurements. ⁇ G° can also be estimated numerically by using the nearest neighbor model as described by SantaLucia, 1998 , Proc Natl Acad Sci USA.
- ITC isothermal titration calorimetry
- oligonucleotides of the present invention hybridize to a target nucleic acid with estimated ⁇ G° values below ⁇ 10 kcal for oligonucleotides that are 10-30 nucleotides in length.
- the degree or strength of hybridization is measured by the standard state Gibbs free energy ⁇ G°.
- the oligonucleotides may hybridize to a target nucleic acid with estimated ⁇ G° values below the range of ⁇ 10 kcal, such as below ⁇ 15 kcal, such as below ⁇ 20 kcal and such as below ⁇ 25 kcal for oligonucleotides that are 8-30 nucleotides in length.
- the oligonucleotides hybridize to a target nucleic acid with an estimated ⁇ G° value of ⁇ 10 to ⁇ 60 kcal, such as ⁇ 12 to ⁇ 40, such as from ⁇ 15 to ⁇ 30 kcal or ⁇ 16 to ⁇ 27 kcal such as ⁇ 18 to ⁇ 25 kcal.
- the target nucleic acid is a nucleic acid which encodes mammalian PD-L1 and may for example be a gene, a RNA, a mRNA, and pre-mRNA, a mature mRNA or a cDNA sequence.
- the target may therefore be referred to as a PD-L1 target nucleic acid.
- the oligonucleotide of the invention may for example target exon regions of a mammalian PD-L1, or may for example target intron region in the PD-L1 pre-mRNA (see Table 1).
- the target nucleic acid encodes a PD-L1 protein, in particular mammalian PD-L1, such as human PD-L1 (See for example tables 2 and 3, which provide reference to the mRNA and pre-mRNA sequences for human, monkey, and mouse PD-L1).
- pre-mRNA is also considered as a nucleic acid that encodes a protein.
- the target nucleic acid is selected from the group consisting of SEQ ID NO: 1, 2 and 3 or naturally occurring variants thereof (e.g. sequences encoding a mammalian PD-L1 protein).
- the target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.
- the oligonucleotide of the invention is typically capable of inhibiting the expression of the PD-L1 target nucleic acid in a cell which is expressing the PD-L1 target nucleic acid.
- the contiguous sequence of nucleobases of the oligonucleotide of the invention is typically complementary to the PD-L1 target nucleic acid, as measured across the length of the oligonucleotide, optionally with the exception of one or two mismatches, and optionally excluding nucleotide based linker regions which may link the oligonucleotide to an optional functional group such as a conjugate, or other non-complementary terminal nucleotides (e.g.
- the target nucleic acid may, in some embodiments, be a RNA or DNA, such as a messenger RNA, such as a mature mRNA or a pre-mRNA.
- the target nucleic acid is a RNA or DNA which encodes mammalian PD-L1 protein, such as human PD-L1, e.g. the human PD-L1 premRNA sequence, such as that disclosed as SEQ ID NO 1 or the human mRNA sequence with NCBI reference number NM_01414. Further information on exemplary target nucleic acids is provided in tables 2 and 3.
- the genome coordinates provide the pre-mRNA sequence (genomic sequence).
- the NCBI reference provides the mRNA sequence (cDNA sequence).
- target sequence refers to a sequence of nucleotides present in the target nucleic acid which comprises the nucleobase sequence which is complementary to the oligonucleotide of the invention.
- the target sequence consists of a region on the target nucleic acid which is complementary to the contiguous nucleotide sequence of the oligonucleotide of the invention.
- the target sequence is longer than the complementary sequence of a single oligonucleotide, and may, for example represent a preferred region of the target nucleic acid which may be targeted by several oligonucleotides of the invention.
- the target sequence may be a sub-sequence of the target nucleic acid.
- the sub-sequence is a sequence selected from the group consisting of a1-a149 (see tables 4). In some embodiments the sub-sequence is a sequence selected from the group consisting of a human PD-L1 mRNA exon, such as a PD-L1 human mRNA exon selected from the group consisting of e1, e2, e3, e4, e5, e6, and e7 (see table 1 above).
- the sub-sequence is a sequence selected from the group consisting of a human PD-L1 mRNA intron, such as a PD-L1 human mRNA intron selected from the group consisting of i1, i2, i3, i4, i5 and i6 (see table 1 above).
- the oligonucleotide of the invention comprises a contiguous nucleotide sequence which is complementary to or hybridizes to the target nucleic acid, such as a sub-sequence of the target nucleic acid, such as a target sequence described herein.
- the oligonucleotide comprises a contiguous nucleotide sequence of at least 8 nucleotides which is complementary to or hybridizes to a target sequence present in the target nucleic acid molecule.
- the contiguous nucleotide sequence (and therefore the target sequence) comprises of at least 8 contiguous nucleotides, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides, such as from 12-25, such as from 14-18 contiguous nucleotides.
- target cell refers to a cell which is expressing the target nucleic acid.
- the target cell may be in vivo or in vitro.
- the target cell is a mammalian cell such as a rodent cell, such as a mouse cell or a rat cell, or a primate cell such as a monkey cell or a human cell.
- the target cell expresses PD-L1 mRNA, such as the PD-L1 pre-mRNA or PD-L1 mature mRNA.
- PD-L1 mRNA such as the PD-L1 pre-mRNA or PD-L1 mature mRNA.
- the poly A tail of PD-L1 mRNA is typically disregarded for antisense oligonucleotide targeting.
- naturally occurring variant refers to variants of PD-L1 gene or transcripts which originate from the same genetic loci as the target nucleic acid, but may differ for example, by virtue of degeneracy of the genetic code causing a multiplicity of codons encoding the same amino acid, or due to alternative splicing of pre-mRNA, or the presence of polymorphisms, such as single nucleotide polymorphisms, and allelic variants. Based on the presence of the sufficient complementary sequence to the oligonucleotide, the oligonucleotide of the invention may therefore target the target nucleic acid and naturally occurring variants thereof.
- the naturally occurring variants have at least 95% such as at least 98% or at least 99% homology to a mammalian PD-L1 target nucleic acid, such as a target nucleic acid selected form the group consisting of SEQ ID NO 1, 2 and 3.
- modulation of expression is to be understood as an overall term for an oligonucleotide's ability to alter the amount of PD-L1 when compared to the amount of PD-L1 before administration of the oligonucleotide.
- modulation of expression may be determined by reference to a control experiment. It is generally understood that the control is an individual or target cell treated with a saline composition or an individual or target cell treated with a non-targeting oligonucleotide (mock). It may however also be an individual treated with the standard of care.
- modulation is an oligonucleotide's ability to inhibit, down-regulate, reduce, suppress, remove, stop, block, prevent, lessen, lower, avoid or terminate expression of PD-L1, e.g. by degradation of mRNA or blockage of transcription.
- modulation is an oligonucleotide's ability to restore, increase or enhance expression of PD-L1, e.g. by repair of splice sites or prevention of splicing or removal or blockage of inhibitory mechanisms such as microRNA repression.
- a high affinity modified nucleoside is a modified nucleotide which, when incorporated into the oligonucleotide enhances the affinity of the oligonucleotide for its complementary target, for example as measured by the melting temperature (T m ).
- a high affinity modified nucleoside of the present invention preferably result in an increase in melting temperature between +0.5 to +12° C., more preferably between +1.5 to +10° C. and most preferably between +3 to +8° C. per modified nucleoside.
- Numerous high affinity modified nucleosides are known in the art and include for example, many 2′ substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).
- the oligomer of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.
- nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.
- Such modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA).
- HNA hexose ring
- LNA ribose ring
- UPA unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons
- Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of
- Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2′-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2′, 3′, 4′ or 5′ positions. Nucleosides with modified sugar moieties also include 2′ modified nucleosides, such as 2′ substituted nucleosides. Indeed, much focus has been spent on developing 2′ substituted nucleosides, and numerous 2′ substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity.
- a 2′ sugar modified nucleoside is a nucleoside which has a substituent other than H or —OH at the 2′ position (2′ substituted nucleoside) or comprises a 2′ linked biradicle, and includes 2′ substituted nucleosides and LNA (2′-4′ biradicle bridged) nucleosides.
- the 2′ modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide.
- 2′ substituted modified nucleosides are 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-RNA, and 2′-F-ANA nucleoside.
- MOE 2′-O-methoxyethyl-RNA
- 2′-amino-DNA 2′-Fluoro-RNA
- 2′-F-ANA nucleoside examples of 2′ substituted modified nucleosides.
- LNA Locked Nucleic Acid Nucleosides
- LNA nucleosides are modified nucleosides which comprise a linker group (referred to as a biradicle or a bridge) between C2′ and C4′ of the ribose sugar ring of a nucleotide. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.
- a linker group referred to as a biradicle or a bridge
- the modified nucleoside or the LNA nucleosides of the oligomer of the invention has a general structure of the formula I or II:
- W is selected from —O—, —S—, —N(R a )—, —C(R a R b )—, such as, in some embodiments —O—;
- B designates a nucleobase or modified nucleobase moiety;
- Z designates an internucleoside linkage to an adjacent nucleoside, or a 5′-terminal group;
- Z* designates an internucleoside linkage to an adjacent nucleoside, or a 3′-terminal group;
- X designates a group selected from the list consisting of —C(R a R b )—, —C(R a ) ⁇ C(R b )—, —C(R a ) ⁇ N—, —O—, —Si(R a ) 2 —, —S—, —SO 2 —, —N(R a )—, and >C ⁇ Z
- the biradicle —X—Y— is —O—CH 2 —
- W is O
- all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- LNA nucleosides are disclosed in WO99/014226, WO00/66604, WO98/039352 and WO2004/046160 which are all hereby incorporated by reference, and include what are commonly known as beta-D-oxy LNA and alpha-L-oxy LNA nucleosides.
- the biradicle —X—Y— is —S—CH 2 —
- W is O
- all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- Such thio LNA nucleosides are disclosed in WO99/014226 and WO2004/046160 which are hereby incorporated by reference.
- the biradicle —X—Y— is —NH—CH 2 —
- W is O
- all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- amino LNA nucleosides are disclosed in WO99/014226 and WO2004/046160 which are hereby incorporated by reference.
- the biradicle —X—Y— is —O—CH 2 —CH 2 — or —O—CH 2 —CH 2 —CH 2 —
- W is O
- all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- LNA nucleosides are disclosed in WO00/047599 and Morita et al, Bioorganic & Med. Chem. Lett. 12 73-76, which are hereby incorporated by reference, and include what are commonly known as 2′-O-4′C-ethylene bridged nucleic acids (ENA).
- the biradicle —X—Y— is —O—CH 2 —
- W is O
- all of R 1 , R 2 , R 3 , and one of R 5 and R 5 * are hydrogen
- the other of R 5 and R 5 * is other than hydrogen such as C 1-6 alkyl, such as methyl.
- the biradicle —X—Y— is —O—CR a R b —, wherein one or both of R a and R b are other than hydrogen, such as methyl, W is O, and all of R 1 , R 2 , R 3 , and one of R 5 and R 5 * are hydrogen, and the other of R 5 and R 5 * is other than hydrogen such as C 1-6 alkyl, such as methyl.
- R a and R b are other than hydrogen, such as methyl
- W is O
- all of R 1 , R 2 , R 3 , and one of R 5 and R 5 * are hydrogen
- the other of R 5 and R 5 * is other than hydrogen such as C 1-6 alkyl, such as methyl.
- the biradicle —X—Y— designate the bivalent linker group —O—CH(CH 2 OCH 3 )— (2′ O-methoxyethyl bicyclic nucleic acid—Seth at al., 2010, J. Org. Chem. Vol 75(5) pp. 1569-81).
- the biradicle —X—Y— designate the bivalent linker group —O—CH(CH 2 CH 3 )— (2′O-ethyl bicyclic nucleic acid—Seth at al., 2010, J. Org. Chem. Vol 75(5) pp. 1569-81).
- the biradicle —X—Y— is —O—CHR a —
- W is O
- all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- 6′ substituted LNA nucleosides are disclosed in WO10036698 and WO07090071 which are both hereby incorporated by reference.
- the biradicle —X—Y— is —O—CH(CH 2 OCH 3 )—, W is O, and all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- LNA nucleosides are also known as cyclic MOEs in the art (cMOE) and are disclosed in WO07090071.
- the biradicle —X—Y— designate the bivalent linker group —O—CH(CH 3 )—. —in either the R- or S-configuration. In some embodiments, the biradicle —X—Y— together designate the bivalent linker group —O—CH 2 —O—CH 2 — (Seth at al., 2010, J. Org. Chem). In some embodiments, the biradicle —X—Y— is —O—CH(CH 3 )—, W is O, and all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- Such 6′ methyl LNA nucleosides are also known as cET nucleosides in the art, and may be either (S)cET or (R)cET stereoisomers, as disclosed in WO07090071 (beta-D) and WO2010/036698 (alpha-L) which are both hereby incorporated by reference).
- the biradicle —X—Y— is —O—CR a R b —, wherein in neither R a or R b is hydrogen, W is O, and all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- R a and R b are both methyl.
- the biradicle —X—Y— is —S—CHR a —
- W is O
- all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- R a is methyl.
- the biradicle —X—Y— is —C( ⁇ CH2)-C(R a R b )—, such as —C( ⁇ CH 2 )—CH 2 —, or —C( ⁇ CH 2 )—CH(CH 3 )—W is O, and all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- vinyl carbo LNA nucleosides are disclosed in WO08154401 and WO09067647 which are both hereby incorporated by reference.
- the biradicle —X—Y— is —N(—OR a )—, W is O, and all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- R a is C 1-6 alkyl such as methyl.
- LNA nucleosides are also known as N substituted LNAs and are disclosed in WO2008/150729, which is hereby incorporated by reference.
- the biradicle —X—Y— together designate the bivalent linker group —O—NR a —CH 3 — (Seth at al., 2010, J. Org. Chem).
- the biradicle —X—Y— is —N(R a )—, W is O, and all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- R a is C 1-6 alkyl such as methyl.
- R 5 and R 5 * is hydrogen and, when substituted the other of R 5 and R 5 * is C 1-6 alkyl such as methyl.
- R 1 , R 2 , R 3 may all be hydrogen, and the biradicle —X—Y— may be selected from —O—CH2- or —O—C(HCR a )—, such as —O—C(HCH3)-.
- the biradicle is —CR a R b —O—CR a R b —, such as CH 2 —O—CH 2 —, W is O and all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- R a is C 1-6 alkyl such as methyl.
- LNA nucleosides are also known as conformationally restricted nucleotides (CRNs) and are disclosed in WO2013036868 which is hereby incorporated by reference.
- the biradicle is —O—CR a R b —O—CR a R b —, such as O—CH 2 —O—CH 2 —, W is O and all of R 1 , R 2 , R 3 , R 5 and R 5 * are all hydrogen.
- R a is C 1-6 alkyl such as methyl.
- LNA nucleosides are also known as COC nucleotides and are disclosed in Mitsuoka et al., Nucleic Acids Research 2009 37(4), 1225-1238, which is hereby incorporated by reference.
- the LNA nucleosides may be in the beta-D or alpha-L stereoisoform.
- the LNA nucleosides in the oligonucleotides are beta-D-oxy-LNA nucleosides.
- Nuclease mediated degradation refers to an oligonucleotide capable of mediating degradation of a complementary nucleotide sequence when forming a duplex with such a sequence.
- the oligonucleotide may function via nuclease mediated degradation of the target nucleic acid, where the oligonucleotides of the invention are capable of recruiting a nuclease, particularly and endonuclease, preferably endoribonuclease (RNase), such as RNase H.
- RNase endoribonuclease
- oligonucleotide designs which operate via nuclease mediated mechanisms are oligonucleotides which typically comprise a region of at least 5 or 6 DNA nucleosides and are flanked on one side or both sides by affinity enhancing nucleosides, for example gapmers, headmers and tailmers.
- the RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule.
- WO01/23613 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH.
- an oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial rate, as measured in pmol/l/min, of at least 5%, such as at least 10% or more than 20% of the of the initial rate determined when using a oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Example 91-95 of WO01/23613 (hereby incorporated by reference).
- gapmer refers to an antisense oligonucleotide which comprises a region of RNase H recruiting oligonucleotides (gap) which is flanked 5′ and 3′ by regions which comprise one or more affinity enhancing modified nucleosides (flanks or wings).
- oligonucleotides capable of recruiting RNase H where one of the flanks is missing, i.e. only one of the ends of the oligonucleotide comprises affinity enhancing modified nucleosides.
- the 3′ flank is missing (i.e. the 5′ flank comprises affinity enhancing modified nucleosides) and for tailmers the 5′ flank is missing (i.e. the 3′ flank comprises affinity enhancing modified nucleosides).
- LNA gapmer is a gapmer oligonucleotide wherein at least one of the affinity enhancing modified nucleosides is an LNA nucleoside.
- mixed wing gapmer or mixed flank gapmer refers to a LNA gapmer wherein at least one of the flank regions comprise at least one LNA nucleoside and at least one non-LNA modified nucleoside, such as at least one 2′ substituted modified nucleoside, such as, for example, 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-RNA and 2′-F-ANA nucleoside(s).
- the mixed wing gapmer has one flank which comprises only LNA nucleosides (e.g. 5′ or 3′) and the other flank (3′ or 5′ respectfully) comprises 2′ substituted modified nucleoside(s) and optionally LNA nucleosides.
- gapbreaker oligonucleotide is used in relation to a gapmer capable of maintaining RNAseH recruitment even though the gap region is disrupted by a non-RNaseH recruiting nucleoside (a gap-breaker nucleoside, E) such that the gap region comprise less than 5 consecutive DNA nucleosides.
- Non-RNaseH recruiting nucleosides are for example nucleosides in the 3′ endo conformation, such as LNA's where the bridge between C2′ and C4′ of the ribose sugar ring of a nucleoside is in the beta conformation, such as beta-D-oxy LNA or ScET nucleoside.
- gapbreaker oligonucleotide to recruit RNaseH is typically sequence or even compound specific—see Rukov et al. 2015 Nucl. Acids Res. Vol. 43 pp. 8476-8487, which discloses “gapbreaker” oligonucleotides which recruit RNaseH which in some instances provide a more specific cleavage of the target RNA.
- the oligonucleotide of the invention is a gapbreaker oligonucleotide.
- the gapbreaker oligonucleotide comprise a 5′-flank (F), a gap (G) and a 3′-flank (F′), wherein the gap is disrupted by a non-RNaseH recruiting nucleoside (a gap-breaker nucleoside, E) such that the gap contain at least 3 or 4 consecutive DNA nucleosides.
- the gapbreaker nucleoside (E) is an LNA nucleoside where the bridge between C2′ and C4′ of the ribose sugar ring of a nucleoside is in the beta conformation and is placed within the gap region such that the gap-breaker LNA nucleoside is flanked 5′ and 3′ by at least 3 (5′) and 3 (3′) or at least 3 (5′) and 4 (3′) or at least 4(5′) and 3(3′) DNA nucleosides, and wherein the oligonucleotide is capable of recruiting RNaseH.
- the gapbreaker oligonucleotide can be represented by the following formulae:
- F-G-E-G-F′ in particular F 1-7 -G 3-4 -E 1 -G 3-4 -F′ 1-7
- D′-F-G-F′ in particular D′ 1-3 -F 1-7 -G 3-4 -E 1 -G 3-4 -F′ 1-7
- F-G-F′-D′′ in particular F 1-7 -G 3-4 -E 1 -G 3-4 -F′ 1-7 -D′′ 1-3
- D′-F-G-F′-D′′ in particular D′ 1-3 -F 1-7 -G 3-4 -E 1 -G 3-4 -F′ 1-7 -D′′ 1-3
- the gapbreaker nucleoside (E) is a beta-D-oxy LNA or ScET or another beta-LNA nucleosides shown in Scheme 1).
- conjugate refers to an oligonucleotide which is covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region), also termed a oligonucleotide conjugate.
- Conjugation of the oligonucleotides of the invention to one or more non-nucleotide moieties may improve the pharmacology of the oligonucleotide, e.g. by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide.
- the conjugate moiety targets the oligonucleotide to the liver. A the same time the conjugate serve to reduce activity of the oligonucleotide in non-target cell types, tissues or organs, e.g. off target activity or activity in non-target cell types, tissues or organs.
- the oligonucleotide conjugate of the invention display improved inhibition of PD-L1 in the target cell when compared to an unconjugated oligonucleotide.
- the oligonucleotide conjugate of the invention has improved cellular distribution between liver and other organs, such as spleen or kidney (i.e. more conjugated oligonucleotide goes to the liver than the spleen or kidney) when compared to an unconjugated oligonucleotide.
- the oligonucleotide conjugate of the invention show improved cellular uptake into the liver of the conjugate oligonucleotide when compared to an unconjugated oligonucleotide.
- WO 93/07883 and WO2013/033230 provides suitable conjugate moieties, which are hereby incorporated by reference. Further suitable conjugate moieties are those capable of binding to the asialoglycoprotein receptor (ASGPr). In particular tri-valent N-acetylgalactosamine conjugate moieties are suitable for binding to the ASGPr, see for example WO 2014/076196, WO 2014/207232 and WO 2014/179620 (hereby incorporated by reference).
- the conjugate moiety is essentially the part of the antisense oligonucleotides conjugates which is not composed of nucleic acids.
- Oligonucleotide conjugates and their synthesis has also been reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S. T. Crooke, ed., Ch. 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, each of which is incorporated herein by reference in its entirety.
- the non-nucleotide moiety is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g. bacterial toxins), vitamins, viral proteins (e.g. capsids) or combinations thereof.
- a linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds.
- Conjugate moieties can be attached to the oligonucleotide directly or through a linking moiety (e.g. linker or tether).
- Linkers serve to covalently connect a third region, e.g. a conjugate moiety (Region C), to a first region, e.g. an oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A).
- the conjugate or oligonucleotide conjugate of the invention may optionally, comprise a linker region (second region or region B and/or region Y) which is positioned between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region).
- a linker region second region or region B and/or region Y
- Region B refers to biocleavable linkers comprising or consisting of a physiologically labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body.
- Conditions under which physiologically labile linkers undergo chemical transformation include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those encountered in mammalian cells.
- Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic enzymes or hydrolytic enzymes or nucleases.
- the biocleavable linker is susceptible to S1 nuclease cleavage.
- the nuclease susceptible linker comprises between 1 and 10 nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides, more preferably between 2 and 6 nucleosides and most preferably between 2 and 4 linked nucleosides comprising at least two consecutive phosphodiester linkages, such as at least 3 or 4 or 5 consecutive phosphodiester linkages.
- the nucleosides are DNA or RNA.
- Phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195 (hereby incorporated by reference).
- Region Y refers to linkers that are not necessarily biocleavable but primarily serve to covalently connect a conjugate moiety (region C or third region), to an oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region).
- the region Y linkers may comprise a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units or amino alkyl groups
- the oligonucleotide conjugates of the present invention can be constructed of the following regional elements A-C, A-B-C, A-B-Y-C, A-Y-B-C or A-Y-C.
- the linker (region Y) is an amino alkyl, such as a C2-C36 amino alkyl group, including, for example C6 to C12 amino alkyl groups. In a preferred embodiment the linker (region Y) is a C6 amino alkyl group.
- treatment refers to both treatment of an existing disease (e.g. a disease or disorder as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognized that treatment as referred to herein may, in some embodiments, be prophylactic.
- the immune response is divided into the innate and adaptive immune response.
- the innate immune system provides an immediate, but non-specific response.
- the adaptive immune response is activated by innate immune response and is highly specific to a particular pathogen.
- immune cells of the adaptive immune response i.e. T and B lymphocytes
- T and B lymphocytes Upon presentation of a pathogen-derived antigen on the surface of antigen-presenting cells, immune cells of the adaptive immune response (i.e. T and B lymphocytes) are activated through their antigen-specific receptors leading to a pathogenic-specific immune response and development of immunological memory.
- Chronic viral infections, such as HBV and HCV are associated with T cell exhaustion characterized by unresponsiveness of the viral-specific T cells. T cell exhaustion is well studied, for a review see for example Yi et al 2010 Immunology 129, 474-481.
- Chronic viral infections are also associated with reduced function of NK cells that are innate immune cells. Enhancing viral immune response is important for clearance of chronic infection. Restoration of immune response against pathogens, mediated by T cells and NK cells, can be assessed by measurement of proliferation, cytokine secretion and cytolytic function (Dolina et al. 2013 Molecular Therapy-Nucleic Acids, 2 e72 and Example 6 herein).
- the present invention relates to the use of antisense oligonucleotides and conjugates thereof and pharmaceutical compositions comprising these to restore immune response against pathogens that have infected an animal, in particular a human.
- the antisense oligonucleotide conjugates of the present invention are particular useful against pathogens that have infected the liver, in particular chronic liver infections like HBV.
- the conjugates allow targeted distribution of the oligonucleotides and prevents systemic knockdown of the target nucleic acid.
- the invention relates to oligonucleotides capable of modulating expression of PD-L1.
- the modulation is may achieved by hybridizing to a target nucleic acid encoding PD-L1 or which is involved in the regulation of PD-L1.
- the target nucleic acid may be a mammalian PD-L1 sequence, such as a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3.
- the target nucleic acid may be a pre-mRNA, an mRNA or any RNA sequence expressed from a mammalian cell that supports the expression or regulation of PD-L1.
- the oligonucleotide of the invention is an antisense oligonucleotide which targets PD-L1.
- the oligonucleotides of the invention are conjugated to a conjugate moiety, in particular an asialoglycoprotein receptor targeting conjugate moiety.
- the antisense oligonucleotide of the invention is capable of modulating the expression of the target by inhibiting or down-regulating it.
- modulation produces an inhibition of expression of at least 20% compared to the normal expression level of the target, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% inhibition compared to the normal expression level of the target.
- such modulation produces an inhibition of expression of at least 20% compared to the expression level when the cell or organism is challenged by an infectious agent, or treated with an agent simulating the challenge by an infectious agent (eg poly I:C or LPS), more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% inhibition compared to the expression level when the cell or organism is challenged by an infectious agent, or treated with an agent simulating the challenge by an infectious agent (eg poly I:C or LPS).
- oligonucleotides of the invention may be capable of inhibiting expression levels of PD-L1 mRNA by at least 60% or 70% in vitro using KARPAS-299 or THP1 cells.
- compounds of the invention may be capable of inhibiting expression levels of PD-L1 protein by at least 50% in vitro using KARPAS-299 or THP1 cells.
- the examples provide assays which may be used to measure PD-L1 RNA (e.g. example 1).
- the target modulation is triggered by the hybridization between a contiguous nucleotide sequence of the oligonucleotide and the target nucleic acid.
- the oligonucleotide of the invention comprises mismatches between the oligonucleotide and the target nucleic acid. Despite mismatches, hybridization to the target nucleic acid may still be sufficient to show a desired modulation of PD-L1 expression.
- Reduced binding affinity resulting from mismatches may advantageously be compensated by increased number of nucleotides in the oligonucleotide and/or an increased number of modified nucleosides capable of increasing the binding affinity to the target, such as 2′ modified nucleosides, including LNA, present within the oligonucleotide sequence.
- the antisense oligonucleotide of the invention is capable of restoring pathogen-specific T cells.
- oligonucleotides of the invention are capable of increasing the pathogen-specific T cells by at least 40%, 50%, 60% or 70% when compared to untreated controls or controls treated with standard of care.
- the antisense oligonucleotide or conjugate of the invention is capable increasing HBV-specific T cells when compared to untreated controls or controls treated with standard of care.
- the examples provide assays which may be used to measure the HBV-specific T cells (e.g. T cell proliferation, cytokine secretion and cytolytic activity).
- the antisense oligonucleotide or conjugate of the invention is capable increasing HCV-specific T cells when compared to untreated controls or controls treated with standard of care. In another embodiment the antisense oligonucleotide or conjugate of the invention is capable increasing HDV-specific T cells when compared to untreated controls or controls treated with standard of care.
- the antisense oligonucleotide of the invention is capable reducing HBsAg levels in an animal or human.
- oligonucleotides of the invention are capable of reducing the HBsAg levels by at least 40%, 50%, 60% or 70%, more preferably by at least 80%, 90% or 95% when compared to the level prior to treatment.
- Most preferably oligonucleotides of the invention are capable of achieving seroconversion of HBsAg in an animal or human infected with HBV.
- An aspect of the present invention relates to an antisense oligonucleotide which comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementarity to a PD-L1 target nucleic acid.
- the oligonucleotide comprises a contiguous sequence which is at least 90% complementary, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid.
- the oligonucleotide of the invention or contiguous nucleotide sequence thereof is fully complementary (100% complementary) to a region of the target nucleic acid, or in some embodiments may comprise one or two mismatches between the oligonucleotide and the target nucleic acid.
- the oligonucleotide comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementary, such as fully (or 100%) complementary, to a region target nucleic acid region present in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments the oligonucleotide sequence is 100% complementary to a corresponding target nucleic acid region present SEQ ID NO: 1 and SEQ ID NO: 2. In some embodiments the oligonucleotide sequence is 100% complementary to a corresponding target nucleic acid region present SEQ ID NO: 1 and SEQ ID NO: 3.
- the oligonucleotide or oligonucleotide conjugate comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementary, such as 100% complementarity, to a corresponding target nucleic acid region wherein the contiguous nucleotide sequence is complementary to a sub-sequence of the target nucleic acid selected from the group consisting of position 371-3068, 5467-12107 and 15317-19511 on SEQ ID NO: 1.
- the sub-sequence of the target nucleic acid is selected from the group consisting of position 371-510, 822-1090, 1992-3068, 5467-5606, 6470-12107, 15317-15720, 15317-18083, 18881-19494 and 1881-19494 on SEQ ID NO: 1.
- the sub-sequence of the target nucleic acid is selected from the group consisting of position 7300-7333, 8028-8072, 9812-9859, 11787-11873 and 15690-15735 on SEQ ID NO: 1.
- the oligonucleotide or oligonucleotide conjugate comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementary, such as 100% complementarity, to a corresponding target nucleic acid region present in SEQ ID NO: 1, wherein the target nucleic acid region is selected from the group consisting of region a1 to a449 in table 4.
- the oligonucleotide or contiguous nucleotide sequence is complementary to a region of the target nucleic acid, wherein the target nucleic acid region is selected from the group consisting of a7, a26, a43, a119, a142, a159, a160, a163, a169, a178, a179, a180, a189, a201, a202, a204, a214, a221, a224, a226, a243, a254, a258, 269, a274, a350, a360, a364, a365, a370, a372, a381, a383, a386, a389, a400, a427, a435 and a438.
- the oligonucleotide or contiguous nucleotide sequence is complementary to a region of the target nucleic acid, wherein the target nucleic acid region is selected from the group consisting of a160, a180, a221, a269 and a360.
- the oligonucleotide of the invention comprises or consists of 8 to 35 nucleotides in length, such as from 9 to 30, such as 10 to 22, such as from 11 to 20, such as from 12 to 18, such as from 13 to 17 or 14 to 16 contiguous nucleotides in length.
- the oligonucleotide comprises or consists of 16 to 20 nucleotides in length. It is to be understood that any range given herein includes the range endpoints. Accordingly, if an oligonucleotide is said to include from 10 to 30 nucleotides, both 10 and 30 nucleotides are included.
- the contiguous nucleotide sequence comprises or consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides in length.
- the oligonucleotide comprises or consists of 16, 17, 18, 19 or 20 nucleotides in length.
- the oligonucleotide or contiguous nucleotide sequence comprises or consists of a sequence selected from the group consisting of sequences listed in table 5.
- the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length with at least 90% identity, preferably 100% identity, to a sequence selected from the group consisting of SEQ ID NO: 5 to 743 (see motif sequences listed in table 5).
- the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length with at least 90% identity, preferably 100% identity, to a sequence selected from the group consisting of SEQ ID NO: 5 to 743 and 771.
- the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length with at least 90% identity, preferably 100% identity, to a sequence selected from the group consisting of SEQ ID NO: 6, 8, 9, 13, 41, 42, 58, 77, 92, 111, 128, 151, 164, 166, 169, 171, 222, 233, 245, 246, 250, 251, 252, 256, 272, 273, 287, 292, 303, 314, 318, 320, 324, 336, 342, 343, 344, 345, 346, 349, 359, 360, 374, 408, 409, 415, 417, 424, 429, 430, 458, 464, 466, 474, 490, 493, 512, 519, 519, 529, 533, 534, 547, 566, 567, 578, 582, 601, 619, 620, 636,
- the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length with at least 90% identity, preferably 100% identity, to SEQ ID NO: 287.
- the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length with at least 90% identity, preferably 100% identity, to SEQ ID NO: 342.
- the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length with at least 90% identity, preferably 100% identity, to SEQ ID NO: 640.
- the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length with at least 90% identity, preferably 100% identity, to SEQ ID NO: 466.
- the antisense oligonucleotide or contiguous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length with at least 90% identity, preferably 100% identity, to SEQ ID NO: 566.
- the motif sequences in table 5 form the contigious nucleotide sequence part of the antisense oligonucleotides of the invention.
- the sequence of the oligonucleotide is equivalent to the contigious nucleotide sequence (e.g. if no biocleavable linkers are added).
- contiguous nucleobase sequences can be modified to for example increase nuclease resistance and/or binding affinity to the target nucleic acid. Modifications are described in the definitions and in the “Oligonucleotide design” section. Table 5 lists preferred designs of each motif sequence.
- Oligonucleotide design refers to the pattern of nucleoside sugar modifications in the oligonucleotide sequence.
- the oligonucleotides of the invention comprise sugar-modified nucleosides and may also comprise DNA or RNA nucleosides.
- the oligonucleotide comprises sugar-modified nucleosides and DNA nucleosides. Incorporation of modified nucleosides into the oligonucleotide of the invention may enhance the affinity of the oligonucleotide for the target nucleic acid.
- the modified nucleosides can be referred to as affinity enhancing modified nucleotides, the modified nucleosides may also be termed units.
- the oligonucleotide comprises at least 1 modified nucleoside, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or at least 16 modified nucleosides.
- the oligonucleotide comprises from 1 to 10 modified nucleosides, such as from 2 to 8 modified nucleosides, such as from 3 to 7 modified nucleosides, such as from 4 to 6 modified nucleosides, such as 3, 4, 5, 6 or 7 modified nucleosides.
- the oligonucleotide comprises one or more sugar modified nucleosides, such as 2′ sugar modified nucleosides.
- the oligonucleotide of the invention comprise the one or more 2′ sugar modified nucleoside independently selected from the group consisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabino nucleic acid (ANA), 2′-fluoro-ANA and LNA nucleosides.
- the one or more modified nucleoside is a locked nucleic acid (LNA).
- the oligonucleotide comprises at least one modified internucleoside linkage.
- all the internucleoside linkages within the contiguous nucleotide sequence are phosphorothioate or boranophosphate internucleoside linkages.
- all the internucleotide linkages in the contiguous sequence of the oligonucleotide are phosphorothioate linkages.
- the oligonucleotide of the invention comprises at least one LNA nucleoside, such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleosides, such as from 2 to 6 LNA nucleosides, such as from 3 to 7 LNA nucleosides, 4 to 6 LNA nucleosides or 3, 4, 5, 6 or 7 LNA nucleosides.
- at least 75% of the modified nucleosides in the oligonucleotide are LNA nucleosides, such as 80%, such as 85%, such as 90% of the modified nucleosides are LNA nucleosides.
- all the modified nucleosides in the oligonucleotide are LNA nucleosides.
- the oligonucleotide may comprise both beta-D-oxy-LNA, and one or more of the following LNA nucleosides: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in either the beta-D or alpha-L configurations or combinations thereof.
- all LNA cytosine units are 5-methyl-cytosine.
- the oligonucleotide or contiguous nucleotide sequence has at least 1 LNA nucleoside at the 5′ end and at least 2 LNA nucleosides at the 3′ end of the nucleotide sequence.
- the oligonucleotide of the invention comprises at least one modified nucleoside which is a 2′-MOE-RNA nucleoside, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2′-MOE-RNA nucleosides.
- at least one of said modified nucleoside is 2′-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2′-fluoro-DNA nucleosides.
- the oligonucleotide of the invention comprises at least one LNA nucleoside and at least one 2′ substituted modified nucleoside.
- the oligonucleotide comprise both 2′ sugar modified nucleosides and DNA units.
- the oligonucleotide comprises both LNA and DNA nucleosides (units).
- the combined total of LNA and DNA units is 8-30, such as 10-25, preferably 12-22, such as 12-18, even more preferably 11-16.
- the nucleotide sequence of the oligonucleotide, such as the contiguous nucleotide sequence consists of at least one or two LNA nucleosides and the remaining nucleosides are DNA units.
- the oligonucleotide comprises only LNA nucleosides and naturally occurring nucleosides (such as RNA or DNA, most preferably DNA nucleosides), optionally with modified internucleoside linkages such as phosphorothioate.
- the oligonucleotide of the invention is capable of recruiting RNase H.
- the structural design of the oligonucleotide of the invention may be selected from gapmers, gapbreakers, headmers and tailmers.
- the oligonucleotide of the invention has a gapmer design or structure also referred herein merely as “Gapmer”.
- Gapmer the oligonucleotide comprises at least three distinct structural regions a 5′-flank, a gap and a 3′-flank, F-G-F′ in ‘5->3’ orientation.
- flanking regions F and F′ (also termed wing regions) comprise a contiguous stretch of modified nucleosides, which are complementary to the PD-L1 target nucleic acid, while the gap region, G, comprises a contiguous stretch of nucleotides which are capable of recruiting a nuclease, preferably an endonuclease such as RNase, for example RNase H, when the oligonucleotide is in duplex with the target nucleic acid.
- Nucleosides which are capable of recruiting a nuclease, in particular RNase H can be selected from the group consisting of DNA, alpha-L-oxy-LNA, 2′-Flouro-ANA and UNA.
- Regions F and F′, flanking the 5′ and 3′ ends of region G preferably comprise non-nuclease recruiting nucleosides (nucleosides with a 3′ endo structure), more preferably one or more affinity enhancing modified nucleosides.
- the 3′ flank comprises at least one LNA nucleoside, preferably at least 2 LNA nucleosides.
- the 5′ flank comprises at least one LNA nucleoside.
- both the 5′ and 3′ flanking regions comprise a LNA nucleoside.
- all the nucleosides in the flanking regions are LNA nucleosides.
- the flanking regions may comprise both LNA nucleosides and other nucleosides (mixed flanks), such as DNA nucleosides and/or non-LNA modified nucleosides, such as 2′ substituted nucleosides.
- the gap is defined as a contiguous sequence of at least 5 RNase H recruiting nucleosides (nucleosides with a 2′ endo structure, preferably DNA) flanked at the 5′ and 3′ end by an affinity enhancing modified nucleoside, preferably LNA, such as beta-D-oxy-LNA. Consequently, the nucleosides of the 5′ flanking region and the 3′ flanking region which are adjacent to the gap region are modified nucleosides, preferably non-nuclease recruiting nucleosides.
- Region F (5′ flank or 5′ wing) attached to the ′5 end of region G comprises, contains or consists of at least one modified nucleoside such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 modified nucleosides.
- region F comprises or consists of from 1 to 7 modified nucleosides, such as from 2 to 6 modified nucleosides, such as from 2 to 5 modified nucleosides, such as from 2 to 4 modified nucleosides, such as from 1 to 3 modified nucleosides, such as 1, 2, 3 or 4 modified nucleosides.
- the F region is defined by having at least on modified nucleoside at the 5′ end and at the 3′ end of the region.
- the modified nucleosides in region F have a 3′ endo structure.
- one or more of the modified nucleosides in region F are 2′ modified nucleosides. In one embodiment all the nucleosides in Region F are 2′ modified nucleosides.
- region F comprises DNA and/or RNA in addition to the 2′ modified nucleosides.
- Flanks comprising DNA and/or RNA are characterized by having a 2′ modified nucleoside in the 5′ end and the 3′ end (adjacent to the G region) of the F region.
- the region F comprise DNA nucleosides, such as from 1 to 3 contiguous DNA nucleosides, such as 1 to 3 or 1 to 2 contiguous DNA nucleosides.
- the DNA nucleosides in the flanks should preferably not be able to recruit RNase H.
- the 2′ modified nucleosides and DNA and/or RNA nucleosides in the F region alternate with 1 to 3 2′ modified nucleosides and 1 to 3 DNA and/or RNA nucleosides.
- Such flanks can also be termed alternating flanks.
- the length of the 5′ flank (region F) in oligonucleotides with alternating flanks may be 4 to 10 nucleosides, such as 4 to 8, such as 4 to 6 nucleosides, such as 4, 5, 6 or 7 modified nucleosides. In some embodiments only the 5′ flank of the oligonucleotide is alternating. Specific examples of region F with alternating nucleosides are
- 2′ indicates a modified nucleoside and N′ is a RNA or DNA.
- all the modified nucleosides in the alternating flanks are LNA and the N′ is DNA.
- one or more of the 2′ modified nucleosides in region F are selected from 2′-O-alkyl-RNA units, 2′-O-methyl-RNA, 2′-amino-DNA units, 2′-fluoro-DNA units, 2′-alkoxy-RNA, MOE units, LNA units, arabino nucleic acid (ANA) units and 2′-fluoro-ANA units.
- the F region comprises both LNA and a 2′ substituted modified nucleoside. These are often termed mixed wing or mixed flank oligonucleotides.
- all the modified nucleosides in region F are LNA nucleosides. In a further embodiment all the nucleosides in Region F are LNA nucleosides. In a further embodiment the LNA nucleosides in region F are independently selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA, cET, and/or ENA, in either the beta-D or alpha-L configurations or combinations thereof. In a preferred embodiment region F comprise at least 1 beta-D-oxy LNA unit, at the 5′ end of the contiguous sequence.
- Region G preferably comprise, contain or consist of at least 4, such as at least 5, such as at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or at least 16 consecutive nucleosides capable of recruiting the aforementioned nuclease, in particular RNaseH.
- region G comprise, contain or consist of from 5 to 12, or from 6 to 10 or from 7 to 9, such as 8 consecutive nucleotide units capable of recruiting aforementioned nuclease.
- the nucleoside units in region G which are capable of recruiting nuclease are in an embodiment selected from the group consisting of DNA, alpha-L-LNA, C4′ alkylated DNA (as described in PCT/EP2009/050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, both incorporated herein by reference), arabinose derived nucleosides like ANA and 2′F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC. 125, 654-661), UNA (unlocked nucleic acid) (as described in Fluiter et al., Mol. Biosyst., 2009, 10, 1039 incorporated herein by reference). UNA is unlocked nucleic acid, typically where the bond between C2 and C3 of the ribose has been removed, forming an unlocked “sugar” residue.
- At least one nucleoside unit in region G is a DNA nucleoside unit, such as from 1 to 18 DNA units, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 DNA units, preferably from 2 to 17 DNA units, such as from 3 to 16 DNA units, such as from 4 to DNA units.
- DNA nucleoside unit such as from 1 to 18 DNA units, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 DNA units, preferably from 2 to 17 DNA units, such as from 3 to 16 DNA units, such as from 4 to DNA units.
- 5 to 14 DNA units such as from 6 to 13 DNA units, such as from 7 to 12 DNA units, such as from 8 to 11 DNA units, more preferably from units 8 to 17 DNA units, or from 9 to 16 DNA units, 10 to 15 DNA units or 11 to 13 DNA units, such as 8, 9, 10, 11, 12, 13, 14, 154, 16, 17 DNA units.
- region G consists of 100% DNA units.
- region G may consist of a mixture of DNA and other nucleosides capable of mediating RNase H cleavage.
- Region G may consist of at least 50% DNA, more preferably 60%, 70% or 80% DNA, and even more preferred 90% or 95% DNA.
- At least one nucleoside unit in region G is an alpha-L-LNA nucleoside unit, such as at least one alpha-L-LNA, such as 2, 3, 4, 5, 6, 7, 8 or 9 alpha-L-LNA.
- region G comprises the least one alpha-L-LNA is alpha-L-oxy-LNA.
- region G comprises a combination of DNA and alpha-L-LNA nucleoside units.
- nucleosides in region G have a 2′ endo structure.
- region G may comprise a gapbreaker nucleoside, leading to a gapbreaker oligonucleotide, which is capable of recruiting RNase H.
- Region F′ (3′ flank or 3′ wing) attached to the ′3 end of region G comprises, contains or consists of at least one modified nucleoside such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 modified nucleosides.
- region F′ comprise or consist of from 1 to 7 modified nucleosides, such as from 2 to 6 modified nucleoside, such as from 2 to 4 modified nucleosides, such as from 1 to 3 modified nucleosides, such as 1, 2, 3 or 4 modified nucleosides.
- the F′ region is defined by having at least on modified nucleoside at the 5′ end and at the 3′ end of the region.
- the modified nucleosides in region F′ have a 3′ endo structure.
- one or more of the modified nucleosides in region F′ are 2′ modified nucleosides. In one embodiment all the nucleosides in Region F′ are 2′ modified nucleosides.
- one or more of the modified nucleosides in region F′ are 2′ modified nucleosides.
- region F′ comprises DNA or RNA in addition to the 2′ modified nucleosides.
- Flanks comprising DNA or RNA are characterized by having a 2′ modified nucleoside in the 5′ end (adjacent to the G region) and the 3′ end of the F′ region.
- region F′ comprises DNA nucleosides, such as from 1 to 4 contiguous DNA nucleosides, such as 1 to 3 or 1 to 2 contiguous DNA nucleosides.
- the DNA nucleosides in the flanks should preferably not be able to recruit RNase H.
- the 2′ modified nucleosides and DNA and/or RNA nucleosides in the F′ region alternate with 1 to 3 2′ modified nucleosides and 1 to 3 DNA and/or RNA nucleosides, such flanks can also be termed alternating flanks.
- the length of the 3′ flank (region F′) in oligonucleotides with alternating flanks may be 4 to 10 nucleosides, such as 4 to 8, such as 4 to 6 nucleosides, such as 4, 5, 6 or 7 modified nucleosides. In some embodiments only the 3′ flank of the oligonucleotide is alternating. Specific examples of region F′ with alternating nucleosides are
- N′ is a RNA or DNA.
- all the modified nucleosides in the alternating flanks are LNA and the N′ is DNA.
- modified nucleosides in region F′ are selected from 2′-O-alkyl-RNA units, 2′-O-methyl-RNA, 2′-amino-DNA units, 2′-fluoro-DNA units, 2′-alkoxy-RNA, MOE units, LNA units, arabino nucleic acid (ANA) units and 2′-fluoro-ANA units.
- the F′ region comprises both LNA and a 2′ substituted modified nucleoside. These are often termed mixed wing or mixed flank oligonucleotides.
- all the modified nucleosides in region F′ are LNA nucleosides. In a further embodiment all the nucleosides in Region F′ are LNA nucleosides. In a further embodiment the LNA nucleosides in region F′ are independently selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA, cET and/or ENA, in either the beta-D or alpha-L configurations or combinations thereof. In a preferred embodiment region F′ has at least 2 beta-D-oxy LNA unit, at the 3′ end of the contiguous sequence.
- Region D′ and D′′ can be attached to the 5′ end of region F or the 3′ end of region F′, respectively. Region D′ or D′′ are optional.
- Region D′ or D′′ may independently comprise 0 to 5, such as 1 to 5, such as 2 to 4, such as 0, 1, 2, 3, 4 or 5 additional nucleotides, which may be complementary or non-complementary to the target nucleic acid.
- the oligonucleotide of the invention may in some embodiments comprise a contiguous nucleotide sequence capable of modulating the target which is flanked at the 5′ and/or 3′ end by additional nucleotides.
- additional nucleotides may serve as a nuclease susceptible biocleavable linker (see definition of linkers).
- the additional 5′ and/or 3′ end nucleosides are linked with phosphodiester linkages, and may be DNA or RNA.
- the additional 5′ and/or 3′ end nucleosides are modified nucleosides which may for example be included to enhance nuclease stability or for ease of synthesis.
- the oligonucleotide of the invention comprises a region D′ and/or D′′ at the 5′ or 3′ end of the contiguous nucleotide sequence.
- the D′ and/or D′′ region is composed of 1 to 5 phosphodiester linked DNA or RNA nucleosides which are not complementary to the target nucleic acid.
- the gapmer oligonucleotide of the present invention can be represented by the following formulae:
- the oligonucleotide conjugates of the present invention have a region C covalently attached to either the 5′ or 3′ end of the oligonucleotide, in particular the gapmer oligonucleotides presented above.
- the oligonucleotide conjugate of the invention comprises a oligonucleotide with the formula 5‘-D’-F-G-F′-3′ or 5‘-F-G-F’-D′′-3′, where region F and F′ independently comprise 1-7 modified nucleosides, G is a region between 6 and 16 nucleosides which are capable of recruiting RNaseH and region D′ or D′′ comprise 1-5 phosphodiester linked nucleosides.
- region D′ or D′′ is present in the end of the oligonucleotide where conjugation to a conjugate moiety is contemplated.
- oligonucleotides with alternating flanks can be represented by the following formulae:
- flank is indicated by F or F′ it only contains 2′ modified nucleosides, such as LNA nucleosides.
- modified nucleosides such as LNA nucleosides.
- the preferred number and types of nucleosides in the alternating regions, and region F, G and F′, D′ and D′′ have been described above.
- the oligonucleotide is a gapmer consisting of 16, 17, 18, 19, 20, 21, 22 nucleotides in length, wherein each of regions F and F′ independently consists of 1, 2, 3 or 4 modified nucleoside units complementary to the PD-L1 target nucleic acid and region G consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 nucleoside units, capable of recruiting nuclease when in duplex with the PD-L1 target nucleic acid and region D′ consists of 2 phosphodiester linked DNAs.
- the oligonucleotide is a gapmer wherein each of regions F and F′ independently consists of 3, 4, 5 or 6 modified nucleoside units, such as nucleoside units containing a 2′-O-methoxyethyl-ribose sugar (2′-MOE) or nucleoside units containing a 2′-fluoro-deoxyribose sugar and/or LNA units, and region G consists of 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 nucleoside units, such as DNA units or other nuclease recruiting nucleosides such as alpha-L-LNA or a mixture of DNA and nuclease recruiting nucleosides.
- each of regions F and F′ independently consists of 3, 4, 5 or 6 modified nucleoside units, such as nucleoside units containing a 2′-O-methoxyethyl-ribose sugar (2′-MOE) or nucleoside units containing a 2′-fluoro-deoxyribo
- the oligonucleotide is a gapmer wherein each of regions F and F′ region consists of two LNA units each, and region G consists of 12, 13, 14 nucleoside units, preferably DNA units.
- Specific gapmer designs of this nature include 2-12-2, 2-13-2 and 2-14-2.
- the oligonucleotide is a gapmer wherein each of regions F and F′ independently consists of three LNA units, and region G consists of 8, 9, 10, 11, 12, 13 or 14 nucleoside units, preferably DNA units.
- Specific gapmer designs of this nature include 3-8-3, 3-9-3 3-10-3, 3-11-3, 3-12-3, 3-13-3 and 3-14-3.
- the oligonucleotide is a gapmer wherein each of regions F and F′ consists of four LNA units each, and region G consists of 8 or 9, 10, 11 or 12 nucleoside units, preferably DNA units.
- Specific gapmer designs of this nature include 4-8-4, 4-9-4, 4-10-4, 4-11-4 and 4-12-4.
- gapmer designs of this nature include F-G-F′ designs selected from a group consisting of a gap with 6 nucleosides and independently 1 to 4 modified nucleosides in the wings including 1-6-1, 1-6-2, 2-6-1, 1-6-3, 3-6-1, 1-6-4, 4-6-1, 2-6-2, 2-6-3, 3-6-2 2-6-4, 4-6-2, 3-6-3, 3-6-4 and 4-6-3 gapmers.
- gapmer designs of this nature include F-G-F′ designs selected from a group consisting of a gap with 7 nucleosides and independently 1 to 4 modified nucleosides in the wings including 1-7-1, 2-7-1, 1-7-2, 1-7-3, 3-7-1, 1-7-4, 4-7-1, 2-7-2, 2-7-3, 3-7-2, 2-7-4, 4-7-2, 3-7-3, 3-7-4, 4-7-3 and 4-7-4 gapmers.
- gapmer designs of this nature include F-G-F′ designs selected from a group consisting of a gap with 8 nucleosides and independently 1 to 4 modified nucleosides in the wings including 1-8-1, 1-8-2, 1-8-3, 3-8-1, 1-8-4, 4-8-1, 2-8-1, 2-8-2, 2-8-3, 3-8-2, 2-8-4, 4-8-2, 3-8-3, 3-8-4, 4-8-3 and 4-8-4 gapmers.
- gapmer designs of this nature include F-G-F′ designs selected from a group consisting of a gap with 9 nucleosides and independently 1 to 4 modified nucleosides in the wings including, 1-9-1, 2-9-1, 1-9-2, 1-9-3, 3-9-1, 1-9-4, 4-9-1, 2-9-2, 2-9-3, 3-9-2, 2-9-4, 4-9-2, 3-9-3, 3-9-4, 4-9-3 and 4-9-4 gapmers.
- gapmer designs of this nature include F-G-F′ designs selected from a group consisting of a gap with 10 nucleosides including, 1-10-1, 2-10-1, 1-10-2, 1-10-3, 3-10-1, 1-10-4, 4-10-1, 2-10-2, 2-10-3, 3-10-2, 2-10-4, 4-10-2, 3-10-3, 3-10-4, 4-10-3 and 4-10-4 gapmers.
- gapmer designs of this nature include F-G-F′ designs selected from a group consisting of a gap with 11 nucleosides including, 1-11-1, 2-11-1, 1-11-2, 1-11-3, 3-11-1, 1-11-4, 4-11-1, 2-11-2, 2-11-3, 3-11-2, 2-11-4, 4-11-2, 3-11-3, 3-11-4, 4-11-3 and 4-11-4 gapmers.
- gapmer designs of this nature include F-G-F′ designs selected from a group consisting of a gap with 12 nucleosides including, 1-12-1, 2-12-1, 1-12-2, 1-12-3, 3-12-1, 1-12-4, 4-12-1, 2-12-2, 2-12-3, 3-12-2, 2-12-4, 4-12-2, 3-12-3, 3-12-4, 4-12-3 and 4-12-4 gapmers.
- gapmer designs of this nature include F-G-F′ designs selected from a group consisting of a gap with 13 nucleosides including, 1-13-1, 2-13-1, 1-13-2, 1-13-3, 3-13-1, 1-13-4, 4-13-1, 2-13-2, 2-13-3, 3-13-2, 2-13-4, 4-13-2, 3-13-3, 3-13-4, 4-13-3 and 4-13-4 gapmers.
- gapmer designs of this nature include F-G-F′ designs selected from a group consisting of a gap with 14 nucleosides including, 1-14-1, 2-14-1, 1-14-2, 1-14-3, 3-14-1, 1-14-4, 4-14-1, 2-14-2, 2-14-3, 3-14-2, 2-14-4, 4-14-2, 3-14-3, 3-14-4, 4-14-3 and 4-14-4 gapmers.
- gapmer designs of this nature include F-G-F′ designs selected from a group consisting of a gap with 15 nucleosides including, 1-15-1, 2-15-1, 1-15-2, 1-15-3, 3-15-1, 1-15-4, 4-15-1, 2-15-2, 2-15-3, 3-15-2, 2-15-4, 4-15-2 and 3-15-3 gapmers.
- gapmer designs of this nature include F-G-F′ designs selected from a group consisting of a gap with 16 nucleosides including, 1-16-1, 2-16-1, 1-16-2, 1-16-3, 3-16-1, 1-16-4, 4-16-1, 2-16-2, 2-16-3, 3-16-2, 2-16-4, 4-16-2 and 3-16-3 gapmers.
- gapmer designs of this nature include F-G-F′ designs selected from a group consisting of a gap with 17 nucleosides including, 1-17-1, 2-17-1, 1-17-2, 1-17-3, 3-17-1, 1-17-4, 4-17-1, 2-17-2, 2-17-3 and 3-17-2 gapmers.
- the F-G-F′ design may further include region D′ and/or D′′, which may have 1, 2 or 3 nucleoside units, such as DNA units, such as 2 phosphodiester linked DNA units.
- region D′ and/or D′′ may have 1, 2 or 3 nucleoside units, such as DNA units, such as 2 phosphodiester linked DNA units.
- the nucleosides in region F and F′ are modified nucleosides, while nucleotides in region G are preferably unmodified nucleosides.
- the preferred modified nucleoside is LNA.
- all the internucleoside linkages in the gap in a gapmer are phosphorothioate and/or boranophosphate linkages. In another embodiment all the internucleoside linkages in the flanks (F and F′ region) in a gapmer are phosphorothioate and/or boranophosphate linkages. In another preferred embodiment all the internucleoside linkages in the D′ and D′′ region in a gapmer are phosphodiester linkages.
- cytosine (C) residues are annotated as 5-methyl-cytosine
- one or more of the Cs present in the oligonucleotide may be unmodified C residues.
- the gapmer is a so-called shortmer as described in WO2008/113832 incorporated herein by reference.
- the oligonucleotide is selected from the group of oligonucleotide compounds with CMP-ID-NO: 5_1 to 743_1 and 771_1.
- the oligonucleotide is selected from the group of oligonucleotide compounds with CMP-ID-NO 6_1, 8_1, 9_1, 13_1, 41_1, 42_1, 58_1, 77_1, 92_1, 111_1, 128_1, 151_1, 164_1, 166_1, 169_1, 171_1, 222_1, 233_1, 245_1, 246_1, 250_1, 251_1, 252_1, 256_1, 272_1, 273_1, 287_1, 292_1, 303_1, 314_1, 318_1, 320_1, 324_1, 336_1, 342_1, 343_1, 344_1, 345_1, 346_1, 349_1, 359_1, 360_1, 374_1, 408_1, 409_1, 415_1, 417_1, 424_1, 429_1, 430_1, 458_1, 464_1, 466_1, 474_1, 490_1, 493_1,
- the oligonucleotide is CMP-ID-NO: 287_1.
- the oligonucleotide is CMP-ID-NO: 342_1.
- the oligonucleotide is CMP-ID-NO: 640_1.
- the oligonucleotide is CMP-ID-NO: 466_1.
- the oligonucleotide is CMP-ID-NO: 566_1.
- the contiguous nucleotide sequence of the oligonucleotide motifs and oligonucleotide compounds of the invention comprise two to four additional phosphodiester linked nucleosides at the 5′ end of the contiguous nucleotide sequence (e.g. region D′).
- the nucleosides serve as a biocleavable linker (see section on biocleavable linkers).
- a ca (cytidine-adenosine) dinucleotide is linked to the 5′ end of contiguous nucleotide sequence (i.e.
- the ca di nucleotide is not complementary to the target sequence at the position where the reminder of the contigious nucleotide is complementary.
- the oligonucleotide or contiguous nucleotide sequence is selected from the group consisting of the nucleotide motif sequences with SEQ ID NO: 766, 767, 768, 769 and 770.
- the oligonucleotide is selected from the group consisting of the oligonucleotide compounds with CMP-ID-NO 766_1, 767_1, 768_1, 769_1 and 770_1.
- Carbohydrate conjugate moieties include but are not limited to galactose, lactose, n-acetylgalactosamine, mannose and mannose-6-phosphate. Carbohydrate conjugates may be used to enhance delivery or activity in a range of tissues, such as liver and/or muscle. See, for example, EP1495769, WO99/65925, Yang et al., Bioconjug Chem (2009) 20(2): 213-21. Zatsepin & Oretskaya Chem Biodivers. (2004) 1(10): 1401-17.
- the carbohydrate conjugate moiety is multivalent, such as, for example 2, 3 or 4 identical or non-identical carbohydrate moieties may be covalently joined to the oligonucleotide, optionally via a linker or linkers.
- the invention provides a conjugate comprising the oligonucleotide of the invention and a carbohydrate conjugate moiety.
- the conjugate moiety is or may comprise mannose or mannose-6-phosphate. This is particular useful for targeting muscle cells, see for example US 2012/122801.
- Conjugate moieties capable of binding to the asialoglycoprotein receptor are particular useful for targeting hepatocytes in liver.
- the invention provides a oligonucleotide conjugate comprising the oligonucleotide of the invention and an asialoglycoprotein receptor targeting conjugate moiety.
- the asialoglycoprotein receptor targeting conjugate moiety comprises one or more carbohydrate moieties capable of binding to the asialoglycoprotein receptor (ASPGr binding carbohydrate moieties) with affinity equal to or greater than that of galactose.
- the affinities of numerous galactose derivatives for the asialoglycoprotein receptor have been studied (see for example: Jobst, S. T. and Drickamer, K. JB.C. 1996, 271, 6686) or are readily determined using methods typical in the art.
- One aspect of the present invention is an antisense oligonucleotide conjugate comprising a) an oligonucleotide (Region A) comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementarity to a PD-L1 target nucleic acid; and b) at least one asialoglycoprotein receptor targeting conjugate moiety (Region C) covalently attached to the oligonucleotide in a).
- the oligonucleotide or a contiguous nucleotide sequence can be as described in any of the sections “oligonucleotides of the invention”, “oligonucleotide design and “gapmer design”.
- asialoglycoprotein receptor targeting conjugate moiety comprises at least one ASPGr binding carbohydrate moiety selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosamine and N-isobutanoylgalactosamine.
- the asialoglycoprotein receptor targeting conjugate moiety is mono-valent, di-valent, tri-valent or tetra-valent (i.e. containing 1, 2, 3 or 4 terminal carbohydrate moieties capable of binding to the asialoglycoprotein receptor).
- the asialoglycoprotein receptor targeting conjugate moiety is di-valent, even more preferred it is trivalent.
- the asialoglycoprotein receptor targeting conjugate moiety comprises 1 to 3 N-acetylgalactosamine (GalNAc) moieties (also termed a GalNAc conjugate).
- the oligonucleotide conjugate comprises a asialoglycoprotein receptor targeting conjugate moiety that is a tri-valent N-acetylgalactosamine (GalNAc) moiety.
- GalNAc conjugates have been used with phosphodiester, methylphosphonate and PNA antisense oligonucleotides (e.g. U.S. Pat. No.
- ASPGr binding carbohydrate moieties are attached to a brancher molecule through the C-I carbons of the saccharides.
- the ASPGr binding carbohydrate moieties are preferably linked to the brancher molecule via spacers.
- a preferred spacer is a flexible hydrophilic spacer (U.S. Pat. No. 5,885,968; Biessen et al. J. Med. Chern. 1995 Vol. 39 p. 1538-1546).
- a preferred flexible hydrophilic spacer is a PEG spacer.
- a preferred PEG spacer is a PEG3 spacer (three ethylene units).
- the brancher molecule can be any small molecule which permits attachment of two or three terminal ASPGr binding carbohydrate moieties and further permits attachment of the branch point to the oligonucleotide.
- An exemplary brancher molecule is a di-lysine.
- a di-lysine molecule contains three amine groups through which three ASPGr binding carbohydrate moieties may be attached and a carboxyl reactive group through which the di-lysine may be attached to the oligonucleotide.
- Alternative brancher molecules may be a doubler or trebler such as those supplied by Glen Research.
- the brancher may be selected from the from the group consisting of 1,3-bis-[5-(4,4′-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)] phosphoramidite (Glen Research Catalogue Number: 10-1920-xx), tris-2,2,2-[3-(4,4′-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research Catalogue Number: 10-1922-xx), tris-2,2,2-[3-(4,4′-dimethoxytrityloxy)propyloxymethyl]methyleneoxypropyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and 1-[5-(4,4′-d
- WO 2014/179620 and PCT application No. PCT/EP2015/073331 describes the generation of various GalNAc conjugate moieties (hereby incorporated by reference).
- One or more linkers may be inserted between the brancher molecule and the oligonucleotide.
- the linker is a biocleavable linker.
- the linker may be selected from the linkers described in the section “Linkers” and its subsections.
- the asialoglycoprotein receptor targeting conjugate moiety in particular the GalNAc conjugate moiety, may be attached to the 3′- or 5′-end of the oligonucleotide using methods known in the art. In preferred embodiments the asialoglycoprotein receptor targeting conjugate moiety is linked to the 5′-end of the oligonucleotide.
- the carbohydrate conjugate moiety comprises a pharmacokinetic modulator selected from the group consisting of a hydrophobic group having 16 or more carbon atoms, hydrophobic group having 16-20 carbon atoms, palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12dienoyl, dioctanoyl, and C16-C20 acyl, and cholesterol.
- the pharmacokinetic modulator containing carbohydrate conjugate moiety is a GalNAc conjugate.
- Preferred carbohydrate conjugate moieties comprises one to three terminal ASPGr binding carbohydrate moieties, preferably N-acetylgalactosamine moiety(s).
- the carbohydrate conjugate moiety comprises three ASPGr binding carbohydrate moieties, preferably N-acetylgalactosamine moieties, linked via a spacer to a brancher molecule.
- the spacer molecule can be between 8 and 30 atoms long.
- a preferred carbohydrate conjugate moiety comprises three terminal GalNAc moieties linked via a PEG spacer to a di-lysine brancher molecule.
- the PEG spacer is a 3PEG spacer.
- Suitable asialoglycoprotein receptor targeting conjugate moieties are shown in FIG. 1 .
- a preferred asialoglycoprotein receptor targeting conjugate moiety is shown in FIG. 3 .
- GalNAc conjugate moieties can include, for example, small peptides with GalNAc moieties attached such as Tyr-Glu-Glu-(aminohexyl GalNAc)3 (YEE(ahGalNAc)3; a glycotripeptide that binds to asialoglycoprotein receptor on hepatocytes, see, e.g., Duff, et al., Methods Enzymol, 2000, 313, 297); lysine-based galactose clusters (e.g., L3G4; Biessen, et al., Cardovasc. Med., 1999, 214); and cholane-based galactose clusters (e.g., carbohydrate recognition motif for asialoglycoprotein receptor).
- small peptides with GalNAc moieties attached such as Tyr-Glu-Glu-(aminohexyl GalNAc)3 (YEE(ahGalNAc)3; a glycotripeptide that binds
- the antisense oligonucleotide conjugate is selected from the group consisting of the following CPM ID NO: 766_2, 767_2, 768_2, 769_2 and 770_2.
- the antisense oligonucleotide conjugate corresponds to the compound represented in FIG. 4 .
- the antisense oligonucleotide conjugate corresponds to the compound represented in FIG. 5 .
- the antisense oligonucleotide conjugate corresponds to the compound represented in FIG. 6 .
- the antisense oligonucleotide conjugate corresponds to the compound represented in FIG. 7 .
- the antisense oligonucleotide conjugate corresponds to the compound represented in FIG. 8 .
- a conjugate is often associated with enhanced pharmacokinetic or pharmeodynamic dynamic properties.
- the presence of a conjugate moiety may interfere with the activity of the oligonucleotide against its intended target, for example via steric hindrance preventing hybridization or nuclease recruitment (e.g. RNAseH).
- a physiologically labile bond biocleavable linker
- region A or first region the oligonucleotide
- the conjugate moiety region C or third region
- Cleavage of the physiologically labile bond occurs spontaneously when a molecule containing the labile bond reaches an appropriate intra- and/or extra-cellular environment.
- a pH labile bond may be cleaved when the molecule enters an acidified endosome.
- a pH labile bond may be considered to be an endosomal cleavable bond.
- Enzyme cleavable bonds may be cleaved when exposed to enzymes such as those present in an endosome or lysosome or in the cytoplasm.
- a disulfide bond may be cleaved when the molecule enters the more reducing environment of the cell cytoplasm.
- a disulfide may be considered to be a cytoplasmic cleavable bond.
- a pH-labile bond is a labile bond that is selectively broken under acidic conditions (pH ⁇ 7). Such bonds may also be termed endosomally labile bonds, since cell endosomes and lysosomes have a pH less than 7.
- the cleavage rate seen in the target tissue is greater than that found in blood serum.
- Suitable methods for determining the level (%) of cleavage in target tissue versus serum or cleavage by S1 nuclease are described in the “Materials and methods” section.
- the biocleavable linker (also referred to as the physiologically labile linker, or nuclease susceptible linker or region B), in a conjugate of the invention, is at least about 20% cleaved, such as at least about 30% cleaved, such as at least about 40% cleaved, such as at least about 50% cleaved, such as at least about 60% cleaved, such as at least about 70% cleaved, such as at least about 75% cleaved when compared against a standard.
- the oligonucleotide conjugate of the invention comprises three regions: i) a first region (region A), which comprises 10-25 contiguous nucleotides complementary to the target nucleic acid; ii) a second region (region B) which comprises a biocleavable linker and iii) a third region (region C) which comprises a conjugate moiety, such as an asialoglycoprotein receptor targeting conjugate moiety, wherein the third region is covalent linked to the second region which is covalently linked to the first region.
- region A a first region
- region B which comprises a biocleavable linker
- region C which comprises a conjugate moiety, such as an asialoglycoprotein receptor targeting conjugate moiety
- the oligonucleotide conjugate comprises a biocleavable linker (Region B) between the contiguous nucleotide sequence (region A) and the asialoglycoprotein receptor targeting conjugate moiety (region C).
- the biocleavable linker may be situated either at the 5′ end and/or the 3′-end of the contiguous nucleotides complementary to the target nucleic acid (region A). In a preferred embodiment the biocleavable linker is at the 5′-end.
- the cleavable linker is susceptible to nuclease(s) which may for example, be expressed in the target cell.
- the biocleavable linker is composed of 2 to 5 consecutive phosphodiester linkages.
- the linker may be a short region (e.g. 1-10 as detailed in the definition of linkers) phosphodiester linked nucleosides.
- the nucleosides in the biocleavable linker region B is (optionally independently) selected from the group consisting of DNA and RNA or modifications thereof which do not interfere with nuclease cleavage.
- Modifications of DNA and RNA nucleosides which do not interfere with nuclease cleavage may be non-naturally occurring nucleobases. Certain sugar-modified nucleosides may also allow nuclease cleavage such as an alpha-L-oxy-LNA.
- all the nucleosides of region B comprise (optionally independently) either a 2′-OH ribose sugar (RNA) or a 2′-H sugar—i.e. RNA or DNA.
- RNA 2′-OH ribose sugar
- RNA or DNA i.e. RNA or DNA.
- at least two consecutive nucleosides of region B are DNA or RNA nucleosides (such as at least 3 or 4 or 5 consecutive DNA or RNA nucleosides).
- the nucleosides of region B are DNA nucleosides
- region B consists of between 1 to 5, or 1 to 4, such as 2, 3, 4 consecutive phosphodiester linked DNA nucleosides.
- region B is so short that it does not recruit RNAseH.
- region B comprises no more than 3 or no more than 4 consecutive phospodiester linked DNA and/or RNA nucleosides (such as DNA nucleosides).
- region A and B may together form the oligonucleotide that is linked to region C.
- region A can be differentiated from region B in that Region A starts with at least one, preferably at least two, modified nucleosides with increased binding affinity to the target nucleic acid (e.g. LNA or nucleosides with a 2′ substituted sugar moiety) and region A on its own is capable of modulation of the expression the target nucleic acid in a relevant cell line.
- region A comprises DNA or RNA nucleosides these are linked with nuclease resistant internucleoside linkage, such phosphorothioate or boranophosphate.
- Region B on the other hand comprises phophodiester linkages between DNA or RNA nucleosides.
- region B is not complementary to or comprises at least 50% mismatches to the target nucleic acid.
- region B is not complementary to the target nucleic acid sequence or to the contiguous nucleotides complementary to the target nucleic acid in region A.
- region B is complementary with the target nucleic acid sequence.
- region A and B together may form a single contiguous sequence which is complementary to the target sequence.
- the internucleoside linkage between the first (region A) and the second region (region B) may be considered part of the second region.
- the sequence of bases in region B is selected to provide an optimal endonuclease cleavage site, based upon the predominant endonuclease cleavage enzymes present in the target tissue or cell or sub-cellular compartment.
- endonuclease cleavage sequences for use in region B may be selected based upon a preferential cleavage activity in the desired target cell (e.g. liver/hepatocytes) as compared to a non-target cell (e.g. kidney).
- the potency of the compound for target down-regulation may be optimized for the desired tissue/cell.
- region B comprises a dinucleotide of sequence AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG, wherein C may be 5-methylcytosine, and/or T may be replaced with U.
- the internucleoside linkage is a phosphodiester linkage.
- region B comprises a trinucleotide of sequence AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG, CGA, CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC, and GGG wherein C may be 5-methylcytosine and/or T may be replaced with U.
- region B comprises a trinucleotide of sequence AAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX, ATGX, ACAX, ACTX, ACCX, ACGX, AGAX, AGTX, AGCX, AGGX, TAAX, TATX, TACX, TAGX, TTAX, TTTX, TTCX, TAGX, TCAX, TCTX, TCCX, TCGX, TGAX, TGTX, TGCX, TGGX, CAAX, CATX, CACX, CAGX, CTAX, CTGX, CTCX, CTTX, CCAX, CCTX, CCCX, CCGX, CGAX, CGTX, CGCX, CGGX, GAAX, GATX, GACX, CAGX, GTAX, GTTX, GTC
- the internucleoside linkages are phosphodiester linkages. It will be recognized that when referring to (naturally occurring) nucleobases A, T, U, G, C, these may be substituted with nucleobase analogues which function as the equivalent natural nucleobase (e.g. base pair with the complementary nucleoside).
- the linker can have at least two functionalities, one for attaching to the oligonucleotide and the other for attaching to the conjugate moiety.
- Example linker functionalities can be electrophilic for reacting with nucleophilic groups on the oligonucleotide or conjugate moiety, or nucleophilic for reacting with electrophilic groups.
- linker functionalities include amino, hydroxyl, carboxylic acid, thiol, phosphoramidate, phosphorothioate, phosphate, phosphite, unsaturations (e.g., double or triple bonds), and the like.
- linkers include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), 6-aminohexyloxy, 4-aminobutyric acid, 4-aminocyclohexylcarboxylic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxy-(6-amido-caproate) (LCSMCC), succinimidyl m-maleimido-benzoylate (MBS), succinimidyl N-e-maleimido-caproylate (EMCS), succinimidyl 6-(beta-maleimido-propionamido) hexanoate (SMPH), succinimidyl N-(a-maleimido acetate
- ADO 8-
- the linker (region Y) is an amino alkyl, such as a C2-C36 amino alkyl group, including, for example C6 to C12 amino alkyl groups. In a preferred embodiment the linker (region Y) is a C6 amino alkyl group.
- the amino alkyl group may be added to the oligonucleotide (region A or region A-B) as part of standard oligonucleotide synthesis, for example using a (e.g. protected) amino alkyl phosphoramidite.
- the linkage group between the amino alkyl and the oligonucleotide may for example be a phosphorothioate or a phosphodiester, or one of the other nucleoside linkage groups referred to herein.
- the amino alkyl group is covalently linked to the 5′ or 3′-end of the oligonucleotide.
- Commercially available amino alkyl linkers are for example 3′-Amino-Modifier reagent for linkage at the 3′-end of the oligonucleotide and for linkage at the 5′-end of an oligonucleotide 5′-Amino-Modifier C6 is available. These reagents are available from Glen Research Corporation (Sterling, Va.).
- Linkers and their use in preparation of conjugates of oligonucleotides are provided throughout the art such as in WO 96/11205 and WO 98/52614 and U.S. Pat. Nos. 4,948,882; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,580,731; 5,486,603; 5,608,046; 4,587,044; 4,667,025; 5,254,469; 5,245,022; 5,112,963; 5,391,723; 5,510475; 5,512,667; 5,574,142; 5,684,142; 5,770,716; 6,096,875; 6,335,432; and 6,335,437, WO 2012/083046 each of which is incorporated by reference in its entirety.
- the invention provides methods for manufacturing the oligonucleotides of the invention comprising reacting nucleotide units and thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide.
- the method uses phophoramidite chemistry (see for example Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313).
- the method further comprises reacting the contiguous nucleotide sequence with a conjugating moiety (ligand).
- composition of the invention comprising mixing the oligonucleotide or conjugated oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
- the invention provides pharmaceutical compositions comprising any of the aforementioned oligonucleotides and/or oligonucleotide conjugates and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
- a pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS) and pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
- the pharmaceutically acceptable diluent is sterile phosphate buffered saline.
- the oligonucleotide is used in the pharmaceutically acceptable diluent at a concentration of 50-300 ⁇ M solution.
- Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).
- WO 2007/031091 provides further suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (hereby incorporated by reference).
- Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in WO2007/031091.
- Oligonucleotides or oligonucleotide conjugates of the invention may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations.
- Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
- compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered.
- the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
- the pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5.
- the resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules.
- composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
- the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug.
- the conjugate moiety is cleaved of the oligonucleotide once the prodrug is delivered to the site of action, e.g. the target cell.
- oligonucleotides or oligonucleotide conjugates of the present invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis.
- oligonucleotides or oligonucleotide conjugates may be used to specifically modulate the synthesis of PD-L1 protein in cells (e.g. in vitro cell cultures) and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention.
- the target modulation is achieved by degrading or inhibiting the mRNA producing the protein, thereby prevent protein formation or by degrading or inhibiting a modulator of the gene or mRNA producing the protein.
- the target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.
- the present invention provides an in vivo or in vitro method for modulating PD-L1 expression in a target cell which is expressing PD-L1, said method comprising administering an oligonucleotide or oligonucleotide conjugate of the invention in an effective amount to said cell.
- the target cell is a mammalian cell in particular a human cell.
- the target cell may be an in vitro cell culture or an in vivo cell forming part of a tissue in a mammal.
- the target cell is present in the liver.
- Liver target cell can be selected from parenchymal cells (e.g. hepatocytes) and non-parenchymal cells such as Kupffer cells, LSECs, stellate cells (or Ito cells), cholangiocytes and liver-associated leukocytes (including T cells and NK cells).
- the target cell is an antigen-presenting cell.
- Antigen-presenting cells displays foreign antigens complexed with major histocompatibility complex (MHC) class I or class II on their surfaces.
- MHC major histocompatibility complex
- the antigen-presenting cell expresses MHC class II (i.e. professional antigen-presenting cells such as dendritic cells, macrophages and B cells).
- the oligonucleotides may be used to detect and quantitate PD-L1 expression in cell and tissues by northern blotting, in-situ hybridisation or similar techniques.
- oligonucleotides or oligonucleotide conjugates of the present invention or pharmaceutical compositions thereof may be administered to an animal or a human, suspected of having a disease or disorder, which can be alleviated or treated by reduction of the expression of PD-L1, in particular by reduction of the expression of PD-L1 in liver target cells.
- the invention provides methods for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.
- the invention also relates to an oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition according to the invention for use as a medicament.
- oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.
- the invention also provides for the use of the oligonucleotide or oligonucleotide conjugate or pharmaceutical composition of the invention as described for the manufacture of a medicament for the treatment of a disease or disorder as referred to herein.
- the disease is selected from a) viral liver infections such as HBV, HCV and HDV; b) parasite infections such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis and c) liver cancer or metastases in the liver.
- the invention relates to oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for use in the treatment of diseases or disorders selected from viral or parasitic infections.
- the disease is selected from a) viral liver infections such as HBV, HCV and HDV; b) parasite infections such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis and c) liver cancer or metastases in the liver.
- disease or disorder is associated with immune exhaustion.
- disease or disorder is associated with exhaustion of virus-specific T-cell responses.
- disease or disorder may be alleviated or treated by reduction of PD-L1 expression.
- the methods of the invention are preferably employed for treatment or prophylaxis against diseases associated with immune exhaustion.
- the oligonucleotide, oligonucleotide conjugate or pharmaceutical compositions of the invention are used in restoration of immune response against a liver cancer or metastases in the liver.
- the oligonucleotide, oligonucleotide conjugate or pharmaceutical compositions of the invention are used in restoration of immune response against a pathogen.
- the pathogen can be found in the liver.
- the pathogens can be a virus or a parasite, in particular those described herein.
- the pathogen is HBV.
- the invention further relates to use of an oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition as defined herein for the manufacture of a medicament for the restoration of immunity against a viral or parasite infection as mentioned herein.
- Oligonucleotides or oligonucleotide conjugates or pharmaceutical compositions of the present invention can be used in the treatment of viral infections, in particular viral infections in the liver where the PD-1 pathway is affected (see for example Kapoor and Kottilil 2014 Future Virol Vol. 9 pp. 565-585 and Salem and EI-Badawy 2015 World J Hepatol Vol. 7 pp. 2449-2458).
- Viral liver infections can be selected from the group consisting of hepatitis viruses, in particular HBV, HCV and HDV, in particular chronic forms of these infections.
- the oligonucleotides or oligonucleotide conjugates or pharmaceutical compositions of the present invention are used to treat HBV, in particular chronic HBV.
- Indicators of chronic HBV infections are high levels of viral load (HBV DNA) and even higher levels of empty HBsAg particles (>100-fold in excess of virions) in the circulation.
- Oligonucleotides or oligonucleotide conjugates of the present invention can also be used to treat viral liver infections that occur as co-infections with HIV.
- Other viral infections which can be treated with the oligonucleotides or oligonucleotide conjugates or pharmaceutical compositions of the present invention are lcmv (Lymphocytic Choriomeningitis Virus), and HIV as a mono infection, HSV-1 and -2, and other herpesviruses. These viruses are not hepatotrophic, however they may be sensitive to PDL1 down regulation.
- the restoration of immunity or immune response involves improvement of the T-cell and/or NK cell response and/or alleviation of the T-cell exhaustion, in particular the HBV-specific T-cell response, the HCV-specific T-cell response and or the HDV-specific T-cell response is restored.
- An improvement of the T cell response can for example be assessed as an increase in T cells in the liver, in particular an increase in CD8+ and/or CD4+ T cells when compared to a control (e.g.
- CD8+ T cells specific for HBV s antigen (HBsAg) and/or CD8+ T cells specific for HBV e antigen (HBeAg) and/or CD8+ T cells specific for HBV core antigen (HBcAg) are increased in subjects treated with an oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the present invention compared to control.
- the HBV antigen specific CD8+ T cells produce one or more cytokines, such as interferon-gamma (IFN- ⁇ ) or tumor necrosis factor alpha (TNF- ⁇ ).
- cytokines such as interferon-gamma (IFN- ⁇ ) or tumor necrosis factor alpha (TNF- ⁇ ).
- IFN- ⁇ interferon-gamma
- TNF- ⁇ tumor necrosis factor alpha
- the increase in CD8+ T cells described above is in particular observed in the liver.
- the increase described herein should be statistically significant when compared to a control.
- the increase is at least 20%, such as 25%, such as 50% such as 75% when compared to control.
- natural killer (NK) cells and/or natural killer T (NKT) cells are activated by the oligonucleotides or oligonucleotide conjugates of the present invention.
- Oligonucleotides or oligonucleotide conjugates or pharmaceutical compositions of the present invention can be used in the treatment parasite infections, in particular parasite infections where the PD-1 pathway is affected (see for example Bhadra et al. 2012 J Infect Dis vol 206 pp. 125-134; Bhadra et al. 2011 Proc Natl Acad Sci USA Vol. 108 pp. 9196-9201; Esch et al. J Immunol vol 191 pp 5542-5550; Freeman and Sharpe 2012 Nat Immunol Vol 13 pp. 113-115; Gutierrez et al. 2011 Infect Immun Vol 79 pp. 1873-1881; Joshi et al.
- Parasite infections can be selected from the group consisting of malaria, toxoplasmosis, leishmaniasis and trypanosomiasis. Malaria infection is caused by protozoa of the genus Plasmodium , in particular of the species P. vivax, P. malariae and P. falciparum . Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii .
- Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania .
- Trypanosomiasis is caused by the protozoan of the genus Trypanosoma .
- Chaga disease which is the tropical form caused by the species Trypanosoma cruzi , and sleeping disease is caused by the species Trypanosoma brucei.
- the restoration of immunity involves restoration of a parasite-specific T cell and NK cell response, in particular a Plasmodium -specific T-cell response, a Toxoplasma gondii -specific T-cell and NK cell response, a Leishmania -specific T-cell and NK cell response, a Trypanosoma cruzi -specific T-cell and NK cell response or a Trypanosoma brucei -specific T-cell and NK cell response.
- a parasite-specific CD8+ T cell and NK cell response that is restored.
- oligonucleotides or pharmaceutical compositions of the present invention may be administered topical (such as, to the skin, inhalation, ophthalmic or otic) or enteral (such as, orally or through the gastrointestinal tract) or parenteral (such as, intravenous, subcutaneous, intra-muscular, intracerebral, intracerebroventricular or intrathecal).
- topical such as, to the skin, inhalation, ophthalmic or otic
- enteral such as, orally or through the gastrointestinal tract
- parenteral such as, intravenous, subcutaneous, intra-muscular, intracerebral, intracerebroventricular or intrathecal.
- the oligonucleotide or pharmaceutical compositions of the present invention are administered by a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g. intracerebral or intraventricular, intravitreal administration.
- a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g. intracerebral or intraventricular, intravitreal administration.
- the active oligonucleotide or oligonucleotide conjugate is administered intravenously.
- the active oligonucleotide or oligonucleotide conjugate is administered subcutaneously.
- the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is administered at a dose of 0.1-15 mg/kg, such as from 0.1-10 mg/kg, such as from 0.2-10 mg/kg, such as from 0.25-10 mg/kg, such as from 0.1-5 mg/kg, such as from 0.2-5 mg/kg, such as from 0.25-5 mg/kg.
- the administration can be once a week, every 2 nd week, every third week or even once a month.
- the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is for use in a combination treatment with another therapeutic agent.
- the therapeutic agent can for example be the standard of care for the diseases or disorders described above.
- the antiviral drugs effective against HBV are for example nucleos(t)ide analogs.
- nucleos(t)ide analogs licensed for therapy of HBV namely lamivudine (Epivir), adefovir (Hepsera), tenofovir (Viread), telbivudine (Tyzeka), entecavir (Baraclude) these are effective in suppressing viral replication (HBV DNA) but have no effect on HBsAg levels.
- Other antiviral drugs include ribavirin and an HBV antibody therapy (monoclonal or polyclonal).
- the immune system modulators can for example be interferon alpha-2a and PEGylated interferon alpha-2a (Pegasys) or TLR7 agonists (e.g. GS-9620) or therapeutic vaccines.
- IFN- ⁇ treatment show only very modest effect in reducing viral load, but result in some HBsAg decline, albeit very inefficiently ( ⁇ 10% after 48 week therapy).
- oligonucleotide or oligonucleotide conjugates of the present invention may also be combined with other antiviral drugs effective against HBV such as the antisense oligonucleotides described in WO2012/145697 and WO 2014/179629 or the siRNA molecules described in WO 2005/014806, WO 2012/024170, WO 2012/2055362, WO 2013/003520 and WO 2013/159109.
- compositions according to the present invention may be comprised of a combination of an oligonucleotide or oligonucleotide conjugate of the present invention in association with a pharmaceutically acceptable excipient, as described herein, and another therapeutic or prophylactic agent known in the art.
- An antisense oligonucleotide which comprises or consists of a contiguous nucleotide sequence of 10 to 30 nucleotides in length capable of reducing the expression of PD-L1.
- oligonucleotide of embodiment 1 or 2 wherein the contiguous nucleotide sequence is complementary to a target nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3.
- oligonucleotide of embodiment 1 to 4 wherein the oligonucleotide is capable of hybridizing to a target nucleic acid of selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3 with a ⁇ G° below ⁇ 10 kcal.
- oligonucleotide of embodiment 1 to 5 wherein the contiguous nucleotide sequence is complementary to a sub-sequence of the target nucleic acid, wherein the sub-sequence is selected from the group consisting of position 371-3068, 5467-12107, 15317-15720, 15317-18083, 15317-19511 and 18881-19494 on SEQ ID NO: 1.
- oligonucleotide of embodiment 6, wherein the sub-sequence is selected from the group consisting of position 7300-7333, 8028-8072, 9812-9859, 11787-11873 and 15690-15735 on SEQ ID NO: 1.
- RNA is mRNA
- oligonucleotide of embodiment 1-10 wherein the contiguous nucleotide sequence comprises or consists of at least 14 contiguous nucleotides, particularly 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 contiguous nucleotides.
- oligonucleotide of embodiment 1-10 wherein the contiguous nucleotide sequence comprises or consists of from 16 to 20 nucleotides.
- oligonucleotide of embodiment 1-10 wherein the oligonucleotide comprises or consists of 14 to 35 nucleotides in length.
- oligonucleotide of embodiment 13 wherein the oligonucleotide comprises or consists of 18 to 22 nucleotides in length.
- oligonucleotide of embodiment 1-15 wherein the contiguous nucleotide sequence is complementary to a sub-sequence of the target nucleic acid, wherein the subsequence is selected from the group consisting of A7, A26, A43, A119, A142, A159, A160, A163, A169, A178, A179, A180, A189, A201, A202, A204, A214, A221, A224, A226, A243, A254, A258, 269, A274, A350, A360, A364, A365, A370, A372, A381, A383, A386, A389, A400, A427, A435 and A438.
- oligonucleotide of embodiment 16 wherein the subsequence is selected from the group consisting of A221, A360, A180, A160 and A269.
- oligonucleotide of embodiment 1-17 wherein the oligonucleotide is not siRNA and is not self-complementary.
- oligonucleotide of embodiment 1-18 wherein the contiguous nucleotide sequence comprises or consists of a sequence selected from SEQ ID NO: 5 to 743 or 771.
- oligonucleotide of embodiment 1-19 wherein the contiguous nucleotide sequence comprises or consists of a sequence selected from SEQ ID NO: 6, 8, 9, 13, 41, 42, 58, 77, 92, 111, 128, 151, 164, 166, 169, 171, 222, 233, 245, 246, 250, 251, 252, 256, 272, 273, 287, 292, 303, 314, 318, 320, 324, 336, 342, 343, 344, 345, 346, 349, 359, 360, 374, 408, 409, 415, 417, 424, 429, 430, 458, 464, 466, 474, 490, 493, 512, 519, 519, 529, 533, 534, 547, 566, 567, 578, 582, 601, 619, 620, 636, 637, 638, 640, 645, 650, 651, 652, 653, 658, 659
- oligonucleotide of embodiment 1-20 wherein the contiguous nucleotide sequence comprises or consists of a sequence selected from SEQ ID NO: 466, 640, 342, 287 and 566.
- oligonucleotide of embodiment 1-25 comprising one or more modified nucleosides.
- oligonucleotide of embodiment 28, wherein the one or more 2′ sugar modified nucleoside is independently selected from the group consisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, 2′-fluoro-ANA and LNA nucleosides.
- modified LNA nucleoside is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA, alpha-L-amino-LNA, beta-D-thio-LNA, alpha-L-thio-LNA, (S)cET, (R)cET beta-D-ENA and alpha-L-ENA.
- oligonucleotide of embodiment 38, wherein the 2′ substituted modified nucleoside is selected from the group consisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-fluoro-DNA, 2′-fluoro-ANA.
- oligonucleotide of embodiment 40 or 41 wherein at least 50% of the internucleoside linkages within the contiguous nucleotide sequence are phosphorothioate internucleoside linkages or boranophosphate internucleoside linkages.
- oligonucleotide of embodiment 44 or 45 wherein the oligonucleotide is a gapmer of formula 5′-F-G-F′-3′, where region F and F′ independently comprise or consist of 1-7 modified nucleosides and G is a region between 6 and 16 nucleosides which are capable of recruiting RNaseH.
- oligonucleotide of embodiment 44 or 45 wherein the gapmer has formula 5′-D′-F-G-F′-3′ or 5′-F-G-F′-D′′-3′, where region F and F′ independently comprise 1-7 modified nucleosides, G is a region between 6 and 16 nucleosides which are capable of recruiting RNaseH and region D′ or D′′ comprise 1-5 phosphodiester linked nucleosides.
- oligonucleotide of embodiment 49, wherein the phosphodiester linked nucleosides are ca (cytidine-adenosine).
- oligonucleotide of embodiment 46 or 47 wherein the modified nucleoside is a 2′ sugar modified nucleoside independently selected from the group consisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabino nucleic acid (ANA), 2′-fluoro-ANA and LNA nucleosides.
- 2′-O-alkyl-RNA 2′-O-methyl-RNA
- 2′-alkoxy-RNA 2′-O-methoxyethyl-RNA
- 2′-amino-DNA 2′-fluoro-DNA
- arabino nucleic acid (ANA) arabino nucleic acid
- 2′-fluoro-ANA and LNA nucleosides arabino nucleic acid
- oligonucleotide of embodiment 52 wherein at least one of region F or F′ further comprises at least one 2′ substituted modified nucleoside independently selected from the group consisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA and 2′-fluoro-DNA.
- oligonucleotide of embodiment 46-56, wherein the RNaseH recruiting nucleosides in region G are independently selected from DNA, alpha-L-LNA, C4′ alkylated DNA, ANA and 2′F-ANA and UNA.
- oligonucleotide of embodiment 57, wherein the nucleosides in region G is DNA and/or alpha-L-LNA nucleosides.
- oligonucleotide of embodiment 1-59 wherein the oligonucleotide is selected from any one of the CMP ID NO: 5_1 to 743_1 and 771_1 (table 5).
- oligonucleotide of embodiment 1-60 wherein the oligonucleotide is selected from the group consisting of CMP ID NO: 6_1, 8_1, 9_1, 13_1, 41_1, 42_1, 58_1, 77_1, 92_1, 111_1, 128_1, 151_1, 164_1, 166_1, 169_1, 171_1, 222_1, 233_1, 245_1, 246_1, 250_1, 251_1, 252_1, 256_1, 272_1, 273_1, 287_1, 292_1, 303_1, 314_1, 318_1, 320_1, 324_1, 336_1, 342_1, 343_1, 344_1, 345_1, 346_1, 349_1, 359_1, 360_1, 374_1, 408_1, 409_1, 415_1, 417_1, 424_1, 429_1, 430_1, 458_1, 464_1, 466_1, 474_1, 490_1, 493
- An antisense oligonucleotide conjugate comprising
- oligonucleotide conjugate of embodiment 63 wherein the conjugate moiety is selected from carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins, vitamins, viral proteins or combinations thereof.
- oligonucleotide conjugate of embodiment 66, wherein the asialoglycoprotein receptor targeting conjugate moiety comprises at least one carbohydrate moiety selected from group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosamine and N-isobutanoylgalactosamine.
- oligonucleotide conjugate of embodiment 66 or 67, wherein the asialoglycoprotein receptor targeting conjugate moiety is mono-valent, di-valent, tri-valent or tetra-valent.
- oligomer conjugate of embodiment 68, wherein the asialoglycoprotein receptor targeting conjugate moiety consists of two to four terminal GalNAc moieties, a PEG spacer linking each GalNAc moiety to a brancher molecule.
- GalNAc N-acetylgalactosamine
- oligonucleotide conjugate of embodiment 63-76 which display improved inhibition of PD-L1 in the target cell, or improved cellular distribution between liver and the spleen or improved cellular uptake into the liver of the conjugate oligonucleotide as compared to an unconjugated oligonucleotide.
- a pharmaceutical composition comprising the oligonucleotide of embodiment 1-62 or a conjugate of embodiment 63-80 and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant.
- a method for manufacturing the oligonucleotide of embodiment 1-62 comprising reacting nucleotide units thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide.
- a method for manufacturing the composition of embodiment 81 comprising mixing the oligonucleotide with a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant.
- a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide of embodiment 1-62 or a conjugate of embodiment 63-80 or the pharmaceutical composition of embodiment 81 to a subject suffering from or susceptible to the disease.
- a method for restoration of immunity against a virus or parasite comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide conjugate of embodiment 63-80 or the oligonucleotide of embodiment 1-62 or the pharmaceutical composition of embodiment 81 to a subject infected with a virus or parasite.
- the restoration of immunity is an increase in the liver of CD8+ T cells specific to one or more HBV antigens when compared to a control.
- oligonucleotide of oligonucleotide of embodiment 1-62 or a conjugate of embodiment 63-80 for the preparation of a medicament for treatment or prevention of a disease in a subject.
- embodiment 91 wherein the restoration of immunity is an increase in the liver of CD8+ T cells specific to one or more HBV antigens when compared to a control.
- Oligonucleotide compounds represent specific designs of a motif sequence.
- Capital letters represent beta-D-oxy LNA nucleosides, lowercase letters represent DNA nucleosides, all LNA C are 5-methyl cytosine, all internucleoside linkages are phosphorothioate internucleoside linkages.
- Oligonucleotides targeting mouse PD-L1 transcript (SEQ ID NO: 4) designs of these, as well as specific oligonucleotide compounds (indicated by CMP ID NO) designed based on the motif sequence.
- SEQ Oligonucleotide CMP Start on SEQ ID NO Motif sequence Design Compound ID NO ID NO: 4 dG 744 agtttacattttctgc 3-10-3 AGTttacattttcTGC 744_1 4189 ⁇ 20 745 tatgtgaagaggagag 3-10-3 TATgtgaagaggaGAG 745_1 7797 ⁇ 19 746 cacctttaaacccca 3-10-3 CACctttaaaaccCCA 746_1 9221 ⁇ 23 747 tcctttataatcacac 3-10-3 TCCtttataatcaCAC 747_1 10386 ⁇ 19 748 acggtatttt
- Oligonucleotide compounds represent specific designs of a motif sequence.
- Capital letters represent beta-D-oxy LNA nucleosides, lowercase letters represent DNA nucleosides, all LNA C are 5-methyl cytosine, all internucleoside linkages are phosphorothioate internucleoside linkages.
- antisense oligonucleotide conjugate CMP ID NO GN2-C6 o c o a o AGTttacattttcTGC 755_2 GN2-C6 o c o a o TATgtgaagaggaGAG 756_2 GN2-C6 o c o a o CACctttaaaaccCCA 757_2 GN2-C6 o c o a o TCCtttataatcaCAC 758_2 GN2-C6 o c o o a o ACGgtattttcacAGG 759_2 GN2-C6 o c o o a o GACactacaatgaGGA 760_2 GN2-C6 o c o a o TGGtttttaggacTGT 761_2 GN2-C6 o c o a o TGGtttttag
- GN2 represents the trivalent GalNAc cluster shown in FIG. 3
- C6 represents an amino alkyl group with 6 carbons
- capital letters represent beta-D-oxy LNA nucleosides
- lowercase letters represent DNA nucleosides
- all LNA C are 5-methyl cytosine
- subscript o represent a phosphodiester nucleoside linkage and unless otherwise indicated internucleoside linkages are phosphorothioate internucleoside linkages.
- Chemical drawings representing some of the molecules are shown in FIGS. 4 to 8 .
- HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice were created and bred at the Institut Pasteur. These mice represent an in vivo experimental model for human immune function studies without any interference with mouse MHC response (Pajot et al 2004 Eur J Immunol. 34(11):3060-9.
- Adeno-associated virus (AAV) vector AAV serotype 2/8 carrying a replication competent HBV DNA genome was used in these studies.
- the AAV-HBV vector (batch GVPN #6163) was diluted in sterile Phosphate buffered Saline (PBS) to reach a titer of 5 ⁇ 10 11 vg/mL.
- Mice were injected intravenously (i.v.) with 100 ⁇ L of this diluted solution (dose/mouse: 5 ⁇ 10 10 vg) in a tail vein.
- Complete viral particles containing HBV DNA were detected in the blood of HBV-carrier mice.
- HBcAg was detected for up to one year in the liver together with HBV circulating proteins HBeAg and HBsAg in the blood.
- HBeAg HBeAg
- HBV DNA persisted in serum for at least one year (Dion et al 2013 J Virol 87:5554-5563).
- mice infected with a recombinant adeno-associated virus (AAV) carrying the HBV genome maintains stable viremia and antigenimia for more than 30 weeks (Dan Yang, et al. 2014 Cellular & Molecular Immunology 11, 71-78).
- mice Male C57BL/6 mice (4-6 weeks old), specific pathogen free, were purchased from SLAC (Shanghai Laboratory Animal Center of Chinese Academy of Sciences) and housed in an animal care facility in individually ventilated cages. Guidelines were followed for the care and use of animals as indicated by WuXi IACUC (Institutional Animal Care and Use Committee, WUXI IACUC protocol number R20131126-Mouse). Mice were allowed to acclimate to the new environment for 3 days and are grouped according to the experimental design.
- Recombinant AAV-HBV was diluted in PBS, 200 ⁇ L per injection. This recombinant virus carries 1.3 copies of the HBV genome (genotype D, serotype ayw).
- mice On day 0, all mice were injected through tail vein with 200 ⁇ L AAV-HBV. On days 6, 13 and 20 after AAV injection, all mice in were submandibularly bled (0.1 ml blood/mouse) for serum collection. On day 22 post injection, mice with stable viremia were ready for oligonucleotide treatment.
- the oligonucleotides can be unconjugated or GalNAc conjugated.
- Plasmid DNA were endotoxin-free and manufactured by Plasmid-Factory (Germany).
- pCMV-S2.S ayw encodes the preS2 and S domains of the HBsAg (genotype D), and its expression is controlled by the cytomegalovirus immediate early gene promoter (Michel et al 1995 Proc Natl Acad Sci USA 92:5307-5311).
- pCMV-HBc encodes the HBV capsid carrying the hepatitis core (HBc) Ag (Dion et al 2013 J Virol 87:5554-5563).
- Treatment with DNA vaccine was conducted as described here. Five days prior to vaccination cardiotoxine (CaTx, Latoxan refL81-02, 50 ⁇ l/muscle) was injected into the muscle of the mice. CaTx depolarizees the muscular fibers to induce cell degeneration, 5 days post injection, new muscular fibers will appear and will receive the DNA vaccine for a better efficacy for transfection.
- cardiotoxine CaTx, Latoxan refL81-02, 50 ⁇ l/muscle
- the antibody was administered by intraperitoneal (i.p.) injection at a dose of 12.5 ⁇ g/g.
- Oligonucleotide synthesis is generally known in the art. Below is a protocol which may be applied. The oligonucleotides of the present invention may have been produced by slightly varying methods in terms of apparatus, support and concentrations used.
- Oligonucleotides are synthesized on uridine universal supports using the phosphoramidite approach on an Oligomaker 48 at 1 ⁇ mol scale. At the end of the synthesis, the oligonucleotides are cleaved from the solid support using aqueous ammonia for 5-16 hours at 60° C. The oligonucleotides are purified by reverse phase HPLC (RP-HPLC) or by solid phase extractions and characterized by UPLC, and the molecular mass is further confirmed by ESI-MS.
- RP-HPLC reverse phase HPLC
- UPLC UPLC
- the coupling of ⁇ -cyanoethyl-phosphoramidites is performed by using a solution of 0.1 M of the 5′-O-DMT-protected amidite in acetonitrile and DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator.
- a phosphoramidite with desired modifications can be used, e.g. a C6 linker for attaching a conjugate group or a conjugate group as such.
- Thiolation for introduction of phosphorthioate linkages is carried out by using xanthane hydride (0.01 M in acetonitrile/pyridine 9:1). Phosphordiester linkages can be introduced using 0.02 M iodine in THF/Pyridine/water 7:2:1. The rest of the reagents are the ones typically used for oligonucleotide synthesis.
- conjugation For post solid phase synthesis conjugation a commercially available C6 amino linker phosphoramidite can be used in the last cycle of the solid phase synthesis and after deprotection and cleavage from the solid support the aminolinked deprotected oligonucleotide is isolated.
- the conjugates are introduced via activation of the functional group using standard synthesis methods.
- the conjugate moiety can be added to the oligonucleotide while still on the solid support by using a GalNAc- or GalNAc-cluster phosphoramidite as described in PCT/EP2015/073331 or in EP appl. NO. 15194811.4.
- the crude compounds are purified by preparative RP-HPLC on a Phenomenex Jupiter 018 10 ⁇ 150 ⁇ 10 mm column. 0.1 M ammonium acetate pH 8 and acetonitrile is used as buffers at a flow rate of 5 mL/min. The collected fractions are lyophilized to give the purified compound typically as a white solid.
- Oligonucleotide and RNA target (phosphate linked, PO) duplexes are diluted to 3 mM in 500 ml RNase-free water and mixed with 500 ml 2 ⁇ T m -buffer (200 mM NaCl, 0.2 mM EDTA, 20 mM Naphosphate, pH 7.0). The solution is heated to 95° C. for 3 min and then allowed to anneal in room temperature for 30 min.
- the duplex melting temperatures (T m ) is measured on a Lambda UV/VIS Spectrophotometer equipped with a Peltier temperature programmer PTP6 using PE Templab software (Perkin Elmer). The temperature is ramped up from 20° C. to 95° C. and then down to 25° C., recording absorption at 260 nm. First derivative and the local maximums of both the melting and annealing are used to assess the duplex T m .
- FAM-labeled oligonucleotides with the biocleavable linker to be tested e.g. a DNA phosphodiester linker (PO linker)
- a DNA phosphodiester linker PO linker
- FAM-labeled oligonucleotides with the biocleavable linker to be tested are subjected to in vitro cleavage using homogenates of the relevant tissues (e.g. liver or kidney) and Serum.
- tissue and serum samples are collected from a suitable animal (e.g. mice, monkey, pig or rat) and homogenized in a homogenisation buffer (0.5% Igepal CA-630, 25 mM Tris pH 8.0, 100 mM NaCl, pH 8.0 (adjusted with 1 N NaOH).
- a homogenisation buffer 0.5% Igepal CA-630, 25 mM Tris pH 8.0, 100 mM NaCl, pH 8.0 (adjusted with 1 N NaOH).
- the tissue homogenates and Serum are spiked with oligonucleotide to concentrations of 200 ⁇ g/g tissue.
- the samples are incubated for 24 hours at 37° C. and thereafter the samples are extracted with phenol-chloroform.
- the solutions are subjected to AIE HPLC analyses on a Dionex Ultimate 3000 using an Dionex DNApac p-100 column and a gradient ranging from 10 mM-1 M sodium perchlorate at pH 7.5.
- the content of cleaved and non-cleaved oligonucleotide are determined against a standard using both a fluorescence detector at 615 nm and a uv detector at 260 nm.
- FAM-labelled oligonucleotides with S1 nuclease susceptible linkers are subjected to in vitro cleavage in S1 nuclease extract or Serum.
- S1 nuclease susceptible linkers e.g. a DNA phosphodiester linker (PO linker)
- oligonucleotides 100 ⁇ M are subjected to in vitro cleavage by S1 nuclease in nuclease buffer (60 U pr. 100 ⁇ L) for 20 and 120 minutes. The enzymatic activity is stopped by adding EDTA to the buffer solution. The solutions are subjected to AIE HPLC analyses on a Dionex Ultimate 3000 using an Dionex DNApac p-100 column and a gradient ranging from 10 mM-1 M sodium perchlorate at pH 7.5. The content of cleaved and non-cleaved oligonucleotide is determined against a standard using both a fluorescence detector at 615 nm and a uv detector at 260 nm.
- Liver cells from AAV/HBV mice were prepared as described below and according to a method described by Tupin et al 2006 Methods Enzymol 417:185-201 with minor modifications. After mouse euthanasia, the liver was perfused with 10 ml of sterile PBS via hepatic portal vein using syringe with G25 needle. When organ is pale, the organ was harvested in Hank's Balanced Salt Solution (HBSS) (GIBCO® HBSS, 24020)+5% decomplemented fetal calf serum (FCS). The harvested liver was gently pressed through 100 ⁇ m cell-strainer (BD Falcon, 352360) and cells were suspended in 30 ml of HBSS+5% FCS.
- HBSS Hank's Balanced Salt Solution
- FCS decomplemented fetal calf serum
- Cells were cultured in complete medium (a-minimal essential medium (Gibco, 22571) supplemented with 10% FCS (Hyclone, # SH30066, lot APG21570), 100 U/mL penicillin+100 ⁇ g/mL streptomycin+0.3 mg/mL L-glutamine (Gibco, 10378), 1 ⁇ non-essential amino acids (Gibco, 11140), 10 mM Hepes (Gibco, 15630), 1 mM sodium pyruvate (Gibco, 11360) and 50 ⁇ M ⁇ -mercaptoethanol (LKB, 1830)).
- FCS Hyclone, # SH30066, lot APG21570
- PBS FACS PBS containing 1% bovine serum albumin and 0.01% sodium azide. Cells were incubated with 5 ⁇ L of PBS FACS containing a rat anti-mouse CD16/CD32 antibody and a viability marker LD fixable yellow, Thermofisher, L34959 for 10 min in the dark at 4° C. Then, cells were stained for 20 min in the dark at 4° C.
- NK P46 BV421 (Rat Mab anti mouse NK P46, Biolegend, 137612) and F4/80 (rat Mab anti-mouse F4/80 FITC, BD Biolegend, 123108) and two supplemental surface markers: PD1 (rat Mab anti-mouse PD1 PE, BD Biosciences, 551892) and PDL1 (rat Mab anti-mouse PDL1 BV711, Biolegend, 124319) were also added.
- Mob monoclonal antibodies against NK P46 BV421
- F4/80 rat Mab anti-mouse F4/80 FITC, BD Biolegend, 123108
- PD1 rat Mab anti-mouse PD1 PE, BD Biosciences, 551892
- PDL1 rat Mab anti-mouse PDL1 BV711, Biolegend, 124319
- ICS assays were performed on both splenocytes and liver mononuclear cells. Cells were seeded in Ubottom 96-well plates. Plates with cells were incubated overnight at 37° C. either in complete medium alone as negative control or with the peptides described in Table 9 at a concentration of 2 ⁇ g/ml. Brefeldin A at 2 ⁇ g/mL (Sigma, B6542) was added after one hour of incubation.
- the mix was composed of monoclonal antibodies against CD3 (hamster Mab anti-mouse CD3-PerCP, BD Biosciences, 553067), CD8 (rat Mab anti-mouse CD8-APC-H7, BD Biosciences, 560182), CD4 (rat Mab anti-mouse CD4-PE-Cy7, BD Biosciences, 552775), and NK cells (Rat Mab anti mouse NK P46 BV421, Biolegend, 137612). Cells were fixed after several washes and permeabilized for 20 min in the dark at room temperature with Cytofix/Cytoperm, washed with Perm/Wash solution (BD Biosciences, 554714) at 4° C.
- CD3 hamster Mab anti-mouse CD3-PerCP, BD Biosciences, 553067
- CD8 rat Mab anti-mouse CD8-APC-H7, BD Biosciences, 560182
- CD4 rat Mab anti-
- Intracellular cytokine staining with antibodies against IFN ⁇ (rat Mab anti-mouse IFN ⁇ -APC, clone XMG1.2, BD Biosciences, 554413) and tumor necrosis factor alpha (TNF ⁇ ) (rat Mab anti-mouse TNF ⁇ -FITC, clone MP6-XT22; 1/250 (BD Biosciences 554418) was performed for 30 min in the dark at 4° C. Before analysis by flow cytometry using the MACSQuant Analyzer, cells were washed with Perm/Wash and re-suspended in PBS FACS containing 1% Formaldehyde.
- Live CD3+CD8+CD4- and cells CD3+CD8-CD4+ were gated and presented on dot-plot. Two regions were defined to gate for positive cells for each cytokine. Numbers of events found in these gates were divided by total number of events in parental population to yield percentages of responding T cells. For each mouse, the percentage obtained in medium alone was considered as background and subtracted from the percentage obtained with peptide stimulations.
- Threshold of positivity was defined according to experiment background i.e. the mean percentage of stained cells obtained for each group in medium alone condition plus two standard deviations. Only percentage of cytokine represented at least 5 events were considered as positive.
- HLA-A2/DR1 restricted epitopes contained in the HBV Core protein and the Envelope domains of the HBsAg (S2 + S).
- a gene walk was performed across the human PD-L1 transcript primarily using 16 to 20mer gapmers. Efficacy testing was performed in an in vitro experiment in the human leukemia monocytic cell line THP1 and in the human non-Hodgkin's K lymphoma cell line (KARPAS-299).
- THP1 and Karpas-299 cell line were originally purchased from European Collection of Authenticated Cell Cultures (ECACC) and maintained as recommended by the supplier in a humidified incubator at 37° C. with 5% CO 2 .
- THP-1 cells (3.104 in RPMI-GLutamax, 10% FBS, 1% Pen-Strep (Thermo Fisher Scientific) were added to the oligonucleotides (4-5 ul) into 96-well round bottom plates and cultured for 6 days in a final volume of 100 ⁇ l/well. Oligonucleotides were screened at one single concentration (20 ⁇ M) and in dose-range concentrations from 25 ⁇ M to 0.004 ⁇ M (1:3 dilution in water). Total mRNA was extracted using the MagNA Pure 96 Cellular RNA Large Volume Kit on the MagNA Pure 96 System (Roche Diagnostics) according to the manufacturer's instructions.
- RT-qPCR was performed using the TaqMan RNA-to-ct 1-Step kit (Thermo Fisher Scientific) on the QuantStudio machine (Applied Biosystems) with pre-designed Taqman primers targeting human PDL1 and ACTB used as endogenous control (Thermo Fisher Scientific).
- the relative PD-L1 mRNA expression level was calculated using 2(-Delta Delta C(T)) method and the percentage of inhibition as the % compared to the control sample (non-treated cells).
- Karpas-299 cells were cultured in RPMI 1640, 2 mM Glutamine and 20% FBS (Sigma). The cells were plated at 10000 cell/well in 96 wells plates incubated for 24 hours before addition of oligonucleotides dissolved in PBS. Final concentration of oligonucleotides was in a single dose of 5 ⁇ M, in a final culture volume was 100 ⁇ l/well or added in a dose response ranging from 50 ⁇ M, 15.8 ⁇ M, 5.0 ⁇ M, 1.58 ⁇ M, 0.5 ⁇ M, 0.158 ⁇ M, 0.05 ⁇ M, to 0.0158 ⁇ M in 100 ⁇ L culture volume.
- cDNA was synthesized using M-MLT Reverse Transcriptase, random decamers RETROscript, RNase inhibitor (Ambion) and 100 mM dNTP set (Invitrogen, PCR Grade) according to the manufacturer's instruction.
- qPCR was performed using TaqMan Fast Advanced Master Mix (2 ⁇ ) (Ambion) in a duplex set up with TaqMan primer assays for the PD-L1 (Applied Biosystems; Hs01125299_m1) and TBP (Applied Biosystems; 4325803).
- the relative PD-L1 mRNA expression level is shown in table 10 as % of control sample (PBS-treated cells).
- KARPAS-299 cells THP1 cells 5 ⁇ M CMP 20 ⁇ M CMP Start on CMP % mRNA % mRNA SEQ ID ID NO of control sd of control sd Compound (CMP) NO 1 4_1 50 1 32 11 TAattggctctacTGC 236 5_1 25 5 9 6 TCGCataagaatgaCT 371 6_1 29 2 15 5 TGaacacacagtcgCA 382 7_1 27 7 3 1 CTGaacacacagtCGC 383 8_1 23 4 11 3 TCTgaacacacagtCG 384 9_1 32 3 19 6 TTCtgaacacacagTC 385 10_1 57 5 39 16 ACaagtcatgttaCTA 463 11_1 75 5 37 12 ACacaagtcatgttAC 465 12_1 22 2 10 3 CTtacttagatgcTGC 495 13_1 33 4 23 11 ACttacttag
- oligonucleotides from Table 10 were tested in KARPAS-299 cells using half-log serial dilutions in PBS (50 ⁇ M, 15.8 ⁇ M, 5.0 ⁇ M, 1.58 ⁇ M, 0.5 ⁇ M, 0.158 ⁇ M, 0.05 ⁇ M, to 0.0158 ⁇ M oligonucleotide) in the in vitro efficacy assay described in Example 1. IC 50 and max inhibition (% residual PD-L1 expression) was assessed for the oligonucleotides.
- EC50 calculations were performed in GraphPad Prism6.
- the IC50 and maximum PD-L1 knock down level is shown in table 11 as % of control (PBS) treated cells.
- oligonucleotides from Table 6 were tested in THP-1 cells using 1:3 serial in water from 25 ⁇ M to 0.004 ⁇ M in the in vitro efficacy assay described in Example 1. IC 50 and max inhibition (Percent residual PD-L1 expression) was assessed for the oligonucleotides.
- EC50 calculations were performed in GraphPad Prism6.
- the IC50 and maximum PD-L1 knock down level is shown in table 12 as % of control (PBS) treated cells.
- MCP-11 cells (originally purchased from ATCC) suspended in DMEM (Sigma cat. no. D0819) supplemented with 10% horse serum, 2 mM L-glutamine, 0.025 mg/ml gentamicin and 1 mM sodium pyruvate were added at a density of 8000 cells/well to the oligonucleotides (10 ⁇ l) in 96-well round bottom plates and cultured for 3 days in a final volume of 200 ⁇ l/well in a humidified incubator at 37° C. with 5% CO 2 .
- Oligonucleotides were screened in dose-range concentrations (50 ⁇ M, 15.8 ⁇ M, 5.0 ⁇ M, 1.58 ⁇ M, 0.5 ⁇ M, 0.158 ⁇ M, 0.05 ⁇ M and 0.0158 ⁇ M).
- qPCR was performed using TaqMan Fast Advanced Master Mix (2 ⁇ ) (Ambion) in a duplex set up with TaqMan primer assays for the PD-L1 (Thermo Fisher Scientific; FAM-MGB Mm00452054-m1) and Gusb (Thermo Fisher Scientific; VIC-MGB-PL Mm01197698-m1).
- the relative PD-L1 mRNA expression level is shown in table 9 as % of residual PD-L1 expression in % of PBS control samples (PBS-treated cells).
- EC50 calculations were performed in GraphPad Prism6.
- the EC50 and maximum PD-L1 knockdown level is shown in table 13 as % of control (PBS) cells.
- mice C57BL/6J female mice (20-23 g; 5 mice per group) were injected s.c. with 5 mg/kg unconjugated oligonucleotides to mouse PD-L1 or 2.8 mg/kg GalNAc-conjugated oligonucleotides to mouse PD-L1.
- mice were injected i.v. with 10 mg/kg poly(I:C) (LWM, Invivogen).
- the mice were sacrificed 5 h after poly(I:C) injection and liver samples were placed in RNA later (Thermo Fisher Scientific) for RNA extraction or frozen at dry ice for protein extraction.
- qPCR was performed using TaqMan® Fast Advanced Master Mix TaqMan Fast Advanced Master Mix (2 ⁇ ) (Ambion) in a duplex set up with TaqMan primer assays for the PD-L1 mRNA (Thermo Fisher Scientific; FAM-MGB Mm00452054-m1) and TBP (Thermo Fisher Scientific; VIC-MGB-PL Mm00446971_m1).
- the relative PD-L1 mRNA expression level is shown in table 13 as % of control samples from mice injected with saline and poly(I:C).
- Liver homogenates were prepared by homogenizing liver samples in 2 ml per 100 mg tissue T-PER® Tissue Protein Extraction Reagent (Thermo Fisher Scientific) mixed with 1 ⁇ Halt Protease Inhibitor Cocktail, EDTA-Free (Thermo Fisher Scientific). Protein concentrations in liver homogenates were measured using Coomassie Plus (Bradford) Assay Reagent (Thermo Scientific) according to the manufacturer's instructions. Liver homogenates (40 ⁇ g protein) were separated on 4-12% Bis-Tris Plus polyacrylamide gels (Thermo Fisher Scientific) in 1 ⁇ MOPS running buffer and transferred to nitrocellulose membranes using iBLOT Dry blotting system (Thermo Fisher Scientific) according to the manufacturer's instructions.
- Each blot was cut in to two parts horizontally at the 64 kDa band.
- the membranes were incubated overnight at 4° C. with rabbit monoclonal anti-vinculin (Abcam cat. no. ab129002) diluted 1:10000 (upper membranes) or goat polyclonal anti-mPD-L1 (R&D Systems cat. no. AF1019) diluted 1:1000 (lower membranes) in TBS containing 5% skim milk and 0.05% Tween20.
- the membranes were washed in TBS containing 0.05% Tween20 and exposed for 1 h at room temperature to HRP-conjugated swine anti-rabbit IgG (DAKO) diluted 1:3000 (upper membranes) or HRP-conjugated rabbit anti-goat IgG (DAKO) diluted 1:2000 in TBS containing 5% skim milk and 0.05% Tween20. Following washing of the membranes, the reactivity was detected using ECL select (Amersham GE Healthcare).
- GalNAc conjugation of the oligonucleotides clearly improves the in vivo PD-L1 reduction.
- the reduction of mRNA generally correlates with a reduction in PD-L1 protein.
- a low in vitro EC50 value generally reflects a good in vivo PD-L1 mRNA reduction once the oligonucleotide is conjugated to GalNAc.
- Example 4 In Vivo PK/PD in Sorted Hepatocytes and Non-Parenchymal Cells from poly(I:C) Induced Mice
- LWM poly(I:C)
- mice were anesthesized 18-20 h after poly(I:C) injection and the liver was perfused at a flow rate of 7 ml per min through the vena cava using Hank's balanced salt solution containing 15 mM Hepes and 0.38 mM EGTA for 5 min followed by collagenase solution (Hank's balanced salt solution containing 0.17 mg/ml Collagenase type 2 (Worthington 4176), 0.03% BSA, 3.2 mM CaCl 2 and 1.6 g/l NaHCO 3 ) for 12 min.
- Hank's balanced salt solution containing 15 mM Hepes and 0.38 mM EGTA
- collagenase solution Hank's balanced salt solution containing 0.17 mg/ml Collagenase type 2 (Worthington 4176), 0.03% BSA, 3.2 mM CaCl 2 and 1.6 g/l NaHCO 3
- Total mRNA was extracted from purified hepatocytes, non-parenchymal cells and total liver suspension (non-fractionated liver cells) using the PureLink Pro 96 RNA Purification kit (Ambion), according to the manufacturer's instructions.
- cDNA was synthesized using M-MLT Reverse Transcriptase, random decamers RETROscript, RNase inhibitor (Ambion) and 100 mM dNTP set (Invitrogen, PCR Grade) according to the manufacturer's instruction.
- qPCR was performed using TaqMan Fast Advanced Master Mix (2 ⁇ ) (Ambion) in a duplex set up with TaqMan primer assays for the PD-L1 (Thermo Fisher Scientific; FAM-MGB Mm00452054-m1) and TBP (Thermo Fisher Scientific; VIC-MGB-PL Mm00446971_m1).
- the relative PD-L1 mRNA expression level is shown in table 10 as % of control samples from mice injected with saline and poly(I:C).
- Oligonucleotide content analysis was performed using ELISA employing a biotinylated capture probe with the sequence 5′-TACCGT-s-Bio-3′ and a digoxigenin conjugated detection probe with the sequence 5′-DIG-C12-S1-CCTGTG-3′.
- the probes consisted of only LNA with a phosphodiester backbone.
- Liver samples (approximately 50 mg) were homogenized in 1.4 mL MagNa pure lysis buffer (Roche Cat. No 03604721001) in a 2 mL Eppendorf tube containing one 5 mm stainless steel bead. Samples were homogenized on Retsch MM400 homogenizer (Merck Eurolab) until a uniform lysate was obtained.
- the homogenized samples were diluted a minimum of 10 times in 5 ⁇ SSCT buffer (750 mM NaCl, and 75 mM sodium citrate, containing 0.05% (v/v) Tween-20, pH 7.0) and a dilution series of 6 times 2 fold dilutions using capture-detection solution (35 nM capture probe and 35 nM detection probe in 5 ⁇ SSCT buffer) were made and incubated for 30 min at room temperature.
- the samples were transferred to a 96 well streptavidin coated plate (Nunc Cat. No. 436014) with 100 ⁇ L in each well. The plates were incubated for 1 hour at room temperature with gentle agitation.
- PD-L1 expression oligo content CMP ID (% of saline-poly(I:C)) (ng/10 5 cells)
- Cell type no Avg SD
- Avg SD Total liver 748_1 31 12.4 2.3 0.3 759_2 28 5.3 8.3 1.1
- AAV/HBV mice were treated with naked or conjugated to GalNAc PD-L1 antisense oligonucleotides, and the PD-L1 mRNA expression and HBV gene expression was evaluated in the liver.
- the mice were transduced by 5 ⁇ 10 10 vg AAV-HBV at week 0 (for further details see description AAV/HBV mouse model in the Materials and Methods section). From W1 post AAV-HBV transduction to W4, mice received 4 additional s.c. injections of PD-L1 oligonucleotides or vehicle (saline solution), given one week apart.
- mice were sacrificed two weeks after the last injections and their liver were removed following PBS perfusion. The liver was cut in smaller pieces and directly frozen.
- F3_core (SEQ ID NO: 784) CTG TGC CTT GGG TGG CTT T Reverse (R3_core): (SEQ ID NO: 785) AAG GAA AGA ACT CAG AAG GCA AAA Probe (P3_core): (SEQ ID NO: 786) 56-FAM-AGC TCC AAA/ZEN/TTC TTT ATA AGG GTC GAT CTC CAT G-3IABkFQ
- a standard curve using HBV plasmid (Genotype D, GTD) was prepared using 10-fold dilutions starting with 1 ⁇ 10 9 copies/ ⁇ l down to 1 copy/ ⁇ l and used in 5 ⁇ l per reaction.
- PD-L1 mRNA expression was measured using qPCR.
- mRNA was extracted from frozen liver pieces that were added to 2 ml tubes containing ceramic beads (Lysing Matrix D tubes, 116913500, mpbio) and 1 ml of Trizol.
- the liver piece was homogenized using the Precellys Tissue Disruptor. 200 ⁇ l Chloroform was added to the homogenate, vortexed and centrifuged at 4° C. for 20 min at 10000 rpm. The RNA containing clear phase (around 500 ul) was transferred into a fresh tube and the same volume of 70% EtOH was added. After mixing well the solution was transferred onto a RNeasy spin column and RNA was further extracted following the RNeasy Kit's manual RNeasy Mini Kit, cat.#74104, Qiagen (including the RNA digestion RNase-free DNase Set, cat.#79254). Elution in 50 ⁇ l H 2 O. The final RNA concentration was measured and adjusted to 100 ng/ul for all samples.
- the qPCR was conducted on 7.5 ⁇ l RNA using the Taqman RNA-to-ct 1-step Kit, cat.#4392938, Thermo Fisher according to the manufactures instructions.
- the fprimer mixed used contained PD-L1_1-3 (Primer number Mm00452054_m1, Mm03048247_m1 and Mm03048248_m1) and endogenous controls (ATCB Mm00607939_s1, CANX Mm00500330_m1, YWHAZ Mm03950126_s1 and GUSB Mm01197698_m1)
- both naked and GalNAc conjugated oligonucleotides are capable of reducing PD-L1 mRNA expression in the liver of an AAV/HBV mouse, with the GalNAc conjugated oligonucleotide being somewhat better. Both oligonucleotides also resulted in some reduction in HBV DNA in the serum.
- AAV/HBV mice from Pasteur were treated with an antibody or antisense oligonucleotides targeting PD-L1.
- the antisense oligonucleotides were either naked or conjugated to GalNAc.
- the animals were immunized with a DNA vaccine against HBs and HBc antigens (see Materials and Methods section) to ensure efficient T cell priming by the antigen presenting cells. It was evaluated how the treatment affected the cell population in liver and spleen, as well as the PD-L1 expression on these populations and whether a HBV specific T cell response could be identified.
- mice Female HLA-A2/DR1 mice were treated according to the protocols below. The study was conducted in two separate sub-studies, with slight differences in the administration regimens as indicated in Table 16 and 17 below.
- DNA vaccine and anti-PD-L1 antibody was administered as described in the materials and method section.
- the antisense oligonucleotides used were CMP ID NO 748_1 (naked) at 5 mg/kg and CMP ID NO: 759_2 (GalNAc conjugated) at 7 mg/kg, both where administered as subcutaneous injections (s.c.).
- liver mononuclear cells of each mouse from each group were collected and depleted of red blood cells (Lysing Buffer, BD biosciences, 555899).
- the liver mononuclear cells required a specific preparation as described in the materials and method section.
- liver mononuclear cells see materials and methods
- PD-L1 protein was evaluated on macrophages, B and T cells from spleen and liver at time of sacrifice.
- the presence of PD-L1 antibody in the surface labeling antibody mix allowed quantification of PD-L1 expressing cells by cytometry.
- PD-L1 was expressed mainly on CD8+ T cells with a mean frequency of 32% and 41% in the control groups (the two vehicle and DNA vaccination groups combined, respectively, FIG. 11A ).
- Treatment with naked PD-L1 oligonucleotide or GalNAc PD-L1 oligonucleotide resulted in a decrease of the frequency of CD8+ T cells expressing PD-L1 (see table 19 FIG. 11A ).
- Significant differences in the % of cells expressing PD-L1 were also noticed for B cells and CD4+ T-cells after ASO treatment, although these cell types express significantly less PD-L1 than the CD8+ T cells (see table 19 and FIGS. 11B and C).
- Treatment with anti-PD-L1 Ab also resulted in an apparent decrease in the PD-L1 expression in all cell types. It is, however, possible that this decrease is due to partly blockage of the PD-L1 epitope by the anti-PD-L1 antibody used for treatment, so that the PD-L1 detection antibody in the surface labeling antibody mix is prevented from binding to PD-L1. Therefore what appears to be a PD-L1 down regulation by the anti-PD-L1 antibody used for treatment may be the result of epitope competition between the treatment antibody and the detection antibody.
- NK cells and CD4+ and CD8+ T cells producing pro-inflammatory cytokines were detected using the intracellular cytokine staining assays (see Materials and Methods section) detecting IFN ⁇ and TNF ⁇ production.
- mice In the spleen no NK cells and few CD4+ T cells secreting IFN ⁇ - and TNF ⁇ were detectable (frequency ⁇ 0.1%) at sacrifice. IFN ⁇ -producing CD8+ T cells targeting the two HBV antigens were detected in mice treated with naked PD-L1 oligonucleotide or GalNAc PD-L1 oligonucleotide as well as in mice from this study which received only DNA vaccine (data not shown).
- IFN ⁇ -secreting CD8+ T cells increased in mice treated with combination of DNA vaccine and naked PD-L1 oligonucleotide or GalNAc PD-L1 oligonucleotide, whereas treatment with anti-PD-L1 antibody did not add any apparent additional effect to the DNA vaccination ( FIG. 12 ).
- IFN ⁇ -producing CD8+ T cells targeting the envelope and core antigens were detected in most DNA-immunized groups (except anti-PD-L1 antibody) ( FIG. 12B ). Most of the S2-S specific T cells produced both IFN ⁇ and TNF ⁇ ( FIG. 12C ). The results are also shown in Table 20.
- mice infected with recombinant adeno-associated virus (AAV) carrying the HBV genome (AAV/HBV) as described under the Shanghai model in the materials and method section were used in this study.
- the mice (6 mice pr. group) were injected once a week for 8 weeks with the antisense oligonucleotide CMP ID NO: 759_2 at 5 mg/kg or vehicle (saline) both where administered as subcutaneous injections (s.c.).
- Blood samples were collected each week during treatment as well as 6 weeks post treatment.
- HBV DNA, HBsAg and HBeAg levels were measured in the serum samples as described below.
- the results for the first 10 weeks are shown in table 21 and in FIG. 13 .
- the study was still ongoing at the time of filing the application therefore data for the remaining 4 weeks have not been obtained.
- Serum HBsAg and HBeAg levels were determined in the serum of infected AAV-HBV mouse using the HBsAg chemoluminescence immunoassay (CLIA) and the HBeAg CLIA kit (Autobio diagnostics Co. Ltd., Zhengzhou, China, Cat. no. CL0310-2 and CL0312-2 respectively), according to the manufacturer's protocol. Briefly, 50 ⁇ l of serum was transferred to the respective antibody coated microtiter plate and 50 ⁇ l of enzyme conjugate reagent was added. The plate was incubated for 60 min on a shaker at room temperature before all wells were washed six times with washing buffer using an automatic washer.
- CLIA chemoluminescence immunoassay
- HBeAg is given in the unit NCU/ml serum.
- mice serum was diluted by a factor of 10 (1:10) with Phosphate buffered saline (PBS).
- DNA was extracted using the MagNA Pure 96 (Roche) robot. 50 ⁇ l of the diluted serum was mixed in a processing cartridge with 200 ul MagNA Pure 96 external lysis buffer (Roche, Cat. no. 06374913001) and incubated for 10 minutes. DNA was then extracted using the “MagNA Pure 96 DNA and Viral Nucleic Acid Small Volume Kit” (Roche, Cat. no. 06543588001) and the “Viral NA Plasma SV external lysis 2.0” protocol. DNA elution volume was 50 ⁇ l.
- GalNAc conjugated PD-L1 antisense oligonucleotide CMP NO 759_2 has a significant effect on the reduction of HBV-DNA, HBsAg and HBeAg levels in serum after 6 weeks of treatment, and effect that is sustained for at least 2 weeks after the treatment has ended.
- GalNAc conjugated PD-L1 antisense oligonucleotide compounds to reduce the PD-L1 transcript in primary human hepatocytes was investigated using genomics.
- Cryopreserved human hepatocytes were suspended in WME supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml) and L-glutamine (0.292 mg/ml) at a density of approx. 5 ⁇ 10 6 cells/ml and seeded into collagen-coated 24-well plates (Becton Dickinson A G, Allschwil, Switzerland) at a density of 2 ⁇ 10 5 cells/well. Cells were pre-cultured for 4 h allowing for attachment to cell culture plates before start of treatment with oligonucleotides at a final concentration of 100 ⁇ M. The oligonucleotides used are shown in table 21 and table 8, vehicle was PBS.
- Seeding medium was replaced by 315 ⁇ l of serum free WME (supplemented with penicillin (100 U/ml), streptomycin (0.1 mg/ml), L-glutamine (0.292 mg/ml)) and 35 ⁇ l of 1 mM oligonucleotide stock solutions in PBS were added to the cell culture and left on the cells for 24 hours or 66 hours.
- serum free WME supplemented with penicillin (100 U/ml), streptomycin (0.1 mg/ml), L-glutamine (0.292 mg/ml)
- 35 ⁇ l of 1 mM oligonucleotide stock solutions in PBS were added to the cell culture and left on the cells for 24 hours or 66 hours.
- Transcript expression profiling was performed using Illumina Stranded mRNA chemistry on the Illumina sequencing platform with a sequencing strategy of 2 ⁇ 51 bp paired end reads and a minimum read depth of 30M per specimen (Q squared EA). Cells were lysed in the wells by adding 350 ⁇ l of Qiagen RLT buffer and were accessioned in a randomization scheme.
- Sequencing libraries were generated for all samples using the Illumina TruSeq Stranded mRNA Library Preparation, starting with 100 ng of total RNA.
- Final cDNA libraries were analyzed for size distribution and using an Agilent Bioanalyzer (DNA 1000 kit), quantitated by qPCR (KAPA Library Quant Kit) and normalized to 2 nM in preparation for sequencing.
- the Standard Cluster Generation Kit v5 was used to bind the cDNA libraries to the flow cell surface and the cBot isothermally to amplify the attached cDNA constructs up to clonal clusters of ⁇ 1000 copies each.
- the DNA sequence was determined by sequencing-by-synthesis technology using the TruSeq SBS Kit.
- Illumina paired-end sequencing reads of length 2 ⁇ 51 bp were mapped on the human reference genome hg19 using the GSNAP short read alignment program. SAM-format alignments were converted into sorted alignment BAM-format files using the SAMTOOLS program. Gene read counts were estimated for PD-L1 based on the exon annotation from NCBI RefSeq, specified by the corresponding GTF file for hg19. A normalization step accounting for the different library size of each sample was applied using the DESeq2 R package.
- n 4 PD-L1 expression PD-L1 expression level 24 h level 66 h (library Compound (library size adjusted counts) size adjusted counts) Vehicle 259 156 159 168 192 136 202 211 767_2 7 7 11 14 22 9 28 15 766_2 16 13 15 10 17 11 29 13 769_2 15 21 18 18 25 18 26 25 768_2 41 25 27 48 31 25 34 22 770_2 21 16 44 62 67 51 38 63
- HepaRG cells (Biopredic International, Saint-Gregoire, France) were cultured in Williams E medium (supplemented with 10% HepaRG growth supplement (Biopredic). From this cell line a HepaRG cell line stably overexpressing human ASGPR1 and ASGPR2 was generated using a lentiviral method. Proliferating HepaRG cells were transduced at MOI 300 with a lentivirus produced on demand by Sirion biotech (CLV-CMV-ASGPR1-T2a_ASGPR2-IRES-Puro) coding for Human ASGPR1 and 2 under the control of a CMV promoter and a puromycin resistance gene.
- Sirion biotech CLV-CMV-ASGPR1-T2a_ASGPR2-IRES-Puro
- Transduced cells were selected for 11 days with 1 ⁇ g/ml puromycin and then maintained in the same concentration of antibiotic to ensure stable expression of the transgenes.
- ASGPR1/2 overexpression was confirmed both at mRNA level by RT-qPCR (ASGPR1: 8560 fold vs non-transduced, ASGPR2: 2389 fold vs non transduced), and at protein level by flow cytometry analysis.
- HBV genotype D was derived from HepG2.2.15 cell culture supernatant and was concentrated using PEG precipitation.
- differentiated ASGPR-HepaRG cells in 96 well plates were infected with HBV at an MOI of 20 to 30 for 20 h, before the cells were washed 4 times with PBS to remove the HBV inoculum.
- RT-qPCR was performed as described in Example 5.
- ActinB was used as the endogenous control to calculate dct values.
- the PD-L1 expression is relative to the endogenous controls and to the saline vehicle.
- PBMCs peripheral blood mononuclear cells
- PBMCs from three chronic HBV infected patients were thawed and seeded at a density of 200,000 cells/well in 100 ⁇ l medium (RPMI1640+GlutaMax+8% Human Serum+25 mM Hepes+1% PenStrep).
- cells were stimulated with 1 ⁇ M PepMix HBV Large Envelope Protein or 1 ⁇ M PepMix HBV Core Protein (see table 9) with or without 5 ⁇ M of CMP ID NO: 466_1 or CMP ID NO: 640_1 in 100 ⁇ l medium containing 100 ⁇ g/ml IL-12 and 5 ng/ml IL-7 (Concanavalin stimulation was only applied at day 8).
- PD-L1 antisense oligonucleotide treatment was renewed with medium containing 50 IU IL-2.
- the cells were re-stimulated with PepMix or 5 ⁇ g/ml Concanavalin A plus PD-L1 antisense oligonucleotide for 24 h.
- 0.1 ⁇ l Brefeldin A, 0.1 ⁇ l Monensin and 3 ⁇ l anti-human CD-107 (APC) were added.
- FACS measurement was performed on a BD Fortessa (BD Biosciences).
- the whole cell population was first gated on live cells (Live and Death stain, BV510), and then on CD3+ (BV605) cells.
- the CD3+ cells were then graphed as CD107a+ (APC) vs IFN ⁇ + (PE).
- the addition of PD-L1 antisense oligonucleotide CMP 466_1 or 640_1 resulted in an additional increase of CD3+ T cell response. This increase was mainly observed in the HBV envelop stimulated group.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Oncology (AREA)
- Epidemiology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Communicable Diseases (AREA)
- Tropical Medicine & Parasitology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
- Marine Sciences & Fisheries (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Rheumatology (AREA)
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/664,749 US10745480B2 (en) | 2016-03-14 | 2019-10-25 | Oligonucleotides for reduction of PD-L1 expression |
US16/839,025 US10829555B2 (en) | 2016-03-14 | 2020-04-02 | Oligonucleotides for reduction of PD-L1 expression |
US17/000,203 US11466081B2 (en) | 2016-03-14 | 2020-08-21 | Oligonucleotides for reduction of PD-L1 expression |
US18/045,109 US20230331837A1 (en) | 2016-03-14 | 2022-10-07 | Oligonucleotides for reduction of pd-l1 expression |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP16160149 | 2016-03-14 | ||
EP16160149.7 | 2016-03-14 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/664,749 Continuation US10745480B2 (en) | 2016-03-14 | 2019-10-25 | Oligonucleotides for reduction of PD-L1 expression |
Publications (1)
Publication Number | Publication Date |
---|---|
US20170283496A1 true US20170283496A1 (en) | 2017-10-05 |
Family
ID=58314191
Family Applications (5)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/458,800 Abandoned US20170283496A1 (en) | 2016-03-14 | 2017-03-14 | Oligonucleotides for reduction of pd-l1 expression |
US16/664,749 Active US10745480B2 (en) | 2016-03-14 | 2019-10-25 | Oligonucleotides for reduction of PD-L1 expression |
US16/839,025 Active US10829555B2 (en) | 2016-03-14 | 2020-04-02 | Oligonucleotides for reduction of PD-L1 expression |
US17/000,203 Active US11466081B2 (en) | 2016-03-14 | 2020-08-21 | Oligonucleotides for reduction of PD-L1 expression |
US18/045,109 Pending US20230331837A1 (en) | 2016-03-14 | 2022-10-07 | Oligonucleotides for reduction of pd-l1 expression |
Family Applications After (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/664,749 Active US10745480B2 (en) | 2016-03-14 | 2019-10-25 | Oligonucleotides for reduction of PD-L1 expression |
US16/839,025 Active US10829555B2 (en) | 2016-03-14 | 2020-04-02 | Oligonucleotides for reduction of PD-L1 expression |
US17/000,203 Active US11466081B2 (en) | 2016-03-14 | 2020-08-21 | Oligonucleotides for reduction of PD-L1 expression |
US18/045,109 Pending US20230331837A1 (en) | 2016-03-14 | 2022-10-07 | Oligonucleotides for reduction of pd-l1 expression |
Country Status (31)
Country | Link |
---|---|
US (5) | US20170283496A1 (zh) |
EP (2) | EP3786297A1 (zh) |
JP (4) | JP6748219B2 (zh) |
KR (4) | KR102580776B1 (zh) |
CN (4) | CN108779465B (zh) |
AR (2) | AR108038A1 (zh) |
AU (3) | AU2017235278C1 (zh) |
BR (1) | BR112018068410A2 (zh) |
CA (2) | CA3120687A1 (zh) |
CL (4) | CL2018002570A1 (zh) |
CO (1) | CO2018007761A2 (zh) |
CR (2) | CR20200119A (zh) |
DK (1) | DK3430141T3 (zh) |
ES (1) | ES2857702T3 (zh) |
HR (1) | HRP20210315T1 (zh) |
HU (1) | HUE053172T2 (zh) |
IL (4) | IL296483B2 (zh) |
LT (1) | LT3430141T (zh) |
MX (2) | MX2018010830A (zh) |
MY (1) | MY194912A (zh) |
PE (3) | PE20181892A1 (zh) |
PH (1) | PH12018501964A1 (zh) |
PL (1) | PL3430141T3 (zh) |
PT (1) | PT3430141T (zh) |
RS (1) | RS61528B1 (zh) |
RU (1) | RU2747822C2 (zh) |
SG (1) | SG11201807854SA (zh) |
SI (1) | SI3430141T1 (zh) |
TW (3) | TWI721128B (zh) |
UA (1) | UA127432C2 (zh) |
WO (1) | WO2017157899A1 (zh) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020081585A1 (en) * | 2018-10-15 | 2020-04-23 | The Brigham And Women's Hospital, Inc. | The long non-coding rna inca1 and homo sapiens heterogeneous nuclear ribonucleoprotein h1 (hnrnph1) as therapeutic targets for immunotherapy |
CN111511914A (zh) * | 2017-10-16 | 2020-08-07 | 豪夫迈·罗氏有限公司 | 减少PAPD5和PAPD7 mRNA的核酸分子用于治疗乙型肝炎感染 |
US10745480B2 (en) | 2016-03-14 | 2020-08-18 | Hoffmann-La Roche, Inc. | Oligonucleotides for reduction of PD-L1 expression |
CN111819283A (zh) * | 2018-01-12 | 2020-10-23 | 百时美施贵宝公司 | 靶向α-突触核蛋白的反义寡核苷酸及其用途 |
US20210214727A1 (en) * | 2019-12-20 | 2021-07-15 | Hoffmann-La Roche Inc. | Enhanced oligonucleotides for inhibiting scn9a expression |
WO2024011109A3 (en) * | 2022-07-06 | 2024-04-11 | Adverum Biotechnologies, Inc. | Compositions and methods for treatment of achromotopsia |
Families Citing this family (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2370451B1 (en) | 2008-12-02 | 2016-11-16 | Wave Life Sciences Japan, Inc. | Method for the synthesis of phosphorus atom modified nucleic acids |
AU2010270714B2 (en) | 2009-07-06 | 2015-08-13 | Wave Life Sciences Ltd. | Novel nucleic acid prodrugs and methods use thereof |
EP2620428B1 (en) | 2010-09-24 | 2019-05-22 | Wave Life Sciences Ltd. | Asymmetric auxiliary group |
JP6128529B2 (ja) | 2011-07-19 | 2017-05-17 | ウェイブ ライフ サイエンシズ リミテッドWave Life Sciences Ltd. | 官能化核酸の合成のための方法 |
CN104661664B (zh) | 2012-07-13 | 2020-07-03 | 波涛生命科学有限公司 | 手性控制 |
EP2872485B1 (en) | 2012-07-13 | 2020-12-16 | Wave Life Sciences Ltd. | Asymmetric auxiliary group |
WO2015108048A1 (ja) | 2014-01-15 | 2015-07-23 | 株式会社新日本科学 | 抗腫瘍作用を有するキラル核酸アジュバンド及び抗腫瘍剤 |
JPWO2015108047A1 (ja) | 2014-01-15 | 2017-03-23 | 株式会社新日本科学 | 免疫誘導活性を有するキラル核酸アジュバンド及び免疫誘導活性剤 |
CA2936712A1 (en) | 2014-01-16 | 2015-07-23 | Meena | Chiral design |
BR112018001232A2 (pt) | 2015-07-21 | 2018-09-25 | The Children's Medical Center Corporation | pd-l1 que expressa células-tronco hematopoéticas e usos |
EP3494219A1 (en) * | 2016-08-03 | 2019-06-12 | Aalborg Universitet | ANTISENSE OLIGONUCLEOTIDES (ASOs) DESIGNED TO INHIBIT IMMUNE CHECKPOINT PROTEINS |
CA3039055A1 (en) | 2016-10-07 | 2018-04-12 | Secarna Pharmaceuticals Gmbh & Co. Kg | Novel approach for treating cancer |
WO2019060708A1 (en) | 2017-09-22 | 2019-03-28 | The Children's Medical Center Corporation | TREATMENT OF TYPE 1 DIABETES AND AUTOIMMUNE DISEASES OR DISORDERS |
WO2020007772A1 (en) * | 2018-07-02 | 2020-01-09 | Roche Innovation Center Copenhagen A/S | Antisense oligonucleotides targeting gbp-1 |
WO2020011653A1 (en) * | 2018-07-09 | 2020-01-16 | Roche Innovation Center Copenhagen A/S | Antisense oligonucleotides targeting kynu |
EP3934695A1 (en) * | 2019-03-05 | 2022-01-12 | F. Hoffmann-La Roche AG | Intracellular targeting of molecules |
CA3132178A1 (en) | 2019-04-02 | 2020-10-08 | Aliye Seda Yilmaz-Elis | Antisense oligonucleotides for immunotherapy |
EP3963074A1 (en) | 2019-05-03 | 2022-03-09 | Secarna Pharmaceuticals GmbH & Co. KG | Pd-l1 antisense oligonucleotides for use in tumor treatment |
JP2023506546A (ja) * | 2019-12-19 | 2023-02-16 | エフ. ホフマン-ラ ロシュ エージー. | B型肝炎ウイルス感染症を処置するためのsept9阻害剤の使用 |
WO2021122910A1 (en) * | 2019-12-19 | 2021-06-24 | F. Hoffmann-La Roche Ag | Use of sbds inhibitors for treating hepatitis b virus infection |
WO2021173812A1 (en) * | 2020-02-28 | 2021-09-02 | Aligos Therapeutics, Inc. | Methods and compositions for targeting pd-l1 |
MX2023001443A (es) | 2020-08-05 | 2023-04-14 | Hoffmann La Roche | Tratamiento con oligonucleotidos de pacientes con hepatitis b. |
WO2023011597A1 (en) * | 2021-08-04 | 2023-02-09 | Hepagene Therapeutics (HK) Limited | Ligand conjugates for delivery of therapeutically active agents |
CN113789324B (zh) * | 2021-08-17 | 2023-08-25 | 广东省大湾区华南理工大学聚集诱导发光高等研究院 | 一种aie探针及其制备方法与在荧光定量pcr方法中的应用 |
AU2022384619A1 (en) | 2021-11-11 | 2024-04-11 | F. Hoffmann-La Roche Ag | Pharmaceutical combinations for treatment of hbv |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010129799A2 (en) * | 2009-05-06 | 2010-11-11 | Curna, Inc. | Treatment of lipid transport and metabolism gene related diseases by inhibition of natural antisense transcript to a lipid transport and metabolism gene |
WO2014118267A1 (en) * | 2013-01-30 | 2014-08-07 | Santaris Pharma A/S | Lna oligonucleotide carbohydrate conjugates |
US20140288153A1 (en) * | 2009-05-28 | 2014-09-25 | Curna, Inc. | Treatment of antiviral gene related diseases by inhibition of natural antisense transcript to an antiviral gene |
Family Cites Families (95)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5927900A (ja) | 1982-08-09 | 1984-02-14 | Wakunaga Seiyaku Kk | 固定化オリゴヌクレオチド |
US4948882A (en) | 1983-02-22 | 1990-08-14 | Syngene, Inc. | Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis |
US4587044A (en) | 1983-09-01 | 1986-05-06 | The Johns Hopkins University | Linkage of proteins to nucleic acids |
US5430136A (en) | 1984-10-16 | 1995-07-04 | Chiron Corporation | Oligonucleotides having selectably cleavable and/or abasic sites |
US5525465A (en) | 1987-10-28 | 1996-06-11 | Howard Florey Institute Of Experimental Physiology And Medicine | Oligonucleotide-polyamide conjugates and methods of production and applications of the same |
DE3738460A1 (de) | 1987-11-12 | 1989-05-24 | Max Planck Gesellschaft | Modifizierte oligonukleotide |
US5391723A (en) | 1989-05-31 | 1995-02-21 | Neorx Corporation | Oligonucleotide conjugates |
US5254469A (en) | 1989-09-12 | 1993-10-19 | Eastman Kodak Company | Oligonucleotide-enzyme conjugate that can be used as a probe in hybridization assays and polymerase chain reaction procedures |
US5486603A (en) | 1990-01-08 | 1996-01-23 | Gilead Sciences, Inc. | Oligonucleotide having enhanced binding affinity |
US5608046A (en) | 1990-07-27 | 1997-03-04 | Isis Pharmaceuticals, Inc. | Conjugated 4'-desmethyl nucleoside analog compounds |
US5245022A (en) | 1990-08-03 | 1993-09-14 | Sterling Drug, Inc. | Exonuclease resistant terminally substituted oligonucleotides |
US5512667A (en) | 1990-08-28 | 1996-04-30 | Reed; Michael W. | Trifunctional intermediates for preparing 3'-tailed oligonucleotides |
RU2123528C1 (ru) * | 1990-11-08 | 1998-12-20 | Чирон Корпорейшн | Способ получения белков hcv, пригодных для использования в вакцине или иммуноанализе, асиалогликопротеин (варианты), композиция для использования в вакцине или в иммуноанализе (варианты), способ очистки асиалогликопротеина и способ понижения содержания или элиминации hcv |
DE69132510T2 (de) | 1990-11-08 | 2001-05-03 | Hybridon Inc | Verbindung von mehrfachreportergruppen auf synthetischen oligonukleotiden |
EP1331011A3 (en) | 1991-10-24 | 2003-12-17 | Isis Pharmaceuticals, Inc. | Derivatized oligonucleotides having improved uptake and other properties |
NL9201440A (nl) | 1992-08-11 | 1994-03-01 | Univ Leiden | Triantennaire clusterglycosiden, hun bereiding en toepassing. |
US5574142A (en) | 1992-12-15 | 1996-11-12 | Microprobe Corporation | Peptide linkers for improved oligonucleotide delivery |
US5580731A (en) | 1994-08-25 | 1996-12-03 | Chiron Corporation | N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith |
WO1996011205A1 (en) | 1994-10-06 | 1996-04-18 | Isis Pharmaceuticals, Inc. | Peptide nucleic acid conjugates |
US5684142A (en) | 1995-06-07 | 1997-11-04 | Oncor, Inc. | Modified nucleotides for nucleic acid labeling |
EP0862439A4 (en) | 1995-11-22 | 2001-01-10 | O Paul O P Ts | LIGANDS FOR INCREASING THE CELLULAR UPtake OF BIOMOLECULES |
JP3756313B2 (ja) | 1997-03-07 | 2006-03-15 | 武 今西 | 新規ビシクロヌクレオシド及びオリゴヌクレオチド類縁体 |
US5770716A (en) | 1997-04-10 | 1998-06-23 | The Perkin-Elmer Corporation | Substituted propargylethoxyamido nucleosides, oligonucleotides and methods for using same |
EP0975370B9 (en) | 1997-05-21 | 2004-11-03 | The Board Of Trustees Of The Leland Stanford Junior University | Composition and method for enhancing transport across biological membranes |
AU9063398A (en) | 1997-09-12 | 1999-04-05 | Exiqon A/S | Oligonucleotide analogues |
US6096875A (en) | 1998-05-29 | 2000-08-01 | The Perlein-Elmer Corporation | Nucleotide compounds including a rigid linker |
US6300319B1 (en) | 1998-06-16 | 2001-10-09 | Isis Pharmaceuticals, Inc. | Targeted oligonucleotide conjugates |
US6335432B1 (en) | 1998-08-07 | 2002-01-01 | Bio-Red Laboratories, Inc. | Structural analogs of amine bases and nucleosides |
US6335437B1 (en) | 1998-09-07 | 2002-01-01 | Isis Pharmaceuticals, Inc. | Methods for the preparation of conjugated oligomers |
TR200604211T1 (tr) | 1999-02-12 | 2007-02-21 | Daiichi Sankyo Company Limiteddaiichi Sankyo Company Limited | Yeni nükleosid ve oligonükleotid analoglarıYeni nükleosid ve oligonükleotid analogları |
EP1178999B1 (en) | 1999-05-04 | 2007-03-14 | Santaris Pharma A/S | L-ribo-lna analogues |
US6617442B1 (en) | 1999-09-30 | 2003-09-09 | Isis Pharmaceuticals, Inc. | Human Rnase H1 and oligonucleotide compositions thereof |
WO2005007855A2 (en) | 2003-07-14 | 2005-01-27 | Sirna Therapeutics, Inc. | RNA INTERFERENCE MEDIATED INHIBITION OF B7-H1 GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA) |
US20060276422A1 (en) | 2001-05-18 | 2006-12-07 | Nassim Usman | RNA interference mediated inhibition of B7-H1 gene expression using short interfering nucleic acid (siNA) |
US20040142325A1 (en) | 2001-09-14 | 2004-07-22 | Liat Mintz | Methods and systems for annotating biomolecular sequences |
EP3287144A1 (en) * | 2002-07-03 | 2018-02-28 | ONO Pharmaceutical Co., Ltd. | Immunopotentiating compositions |
DK2141233T3 (en) | 2002-11-18 | 2017-01-09 | Roche Innovation Ct Copenhagen As | Antisense Design |
JP4980716B2 (ja) | 2003-06-12 | 2012-07-18 | アルナイラム ファーマシューティカルズ, インコーポレイテッド | 遺伝子サイレンシングに有用な保存されたhbv及びhcv配列 |
DK1495769T3 (da) | 2003-07-11 | 2008-06-23 | Lbr Medbiotech B V | Mannose-6-phosphat-receptormedieret gentransfer til muskelceller |
PL1810026T3 (pl) * | 2004-10-06 | 2018-08-31 | Mayo Foundation For Medical Education And Research | B7-H1 i PD-1 w leczeniu raka nerkowokomórkowego |
ZA200703482B (en) * | 2004-10-06 | 2008-09-25 | Mayo Foundation | B7-H1 and methods of diagnosis, prognosis, and treatment of cancer |
US20120122801A1 (en) | 2005-01-05 | 2012-05-17 | Prosensa B.V. | Mannose-6-phosphate receptor mediated gene transfer into muscle cells |
HUE030877T2 (en) | 2005-06-08 | 2017-06-28 | Dana Farber Cancer Inst Inc | Methods and Compositions for the Treatment of Permanent Infections and Cancer by Inhibition of Programmed Cell Death (PD-1) Synthesis |
WO2007031091A2 (en) | 2005-09-15 | 2007-03-22 | Santaris Pharma A/S | Rna antagonist compounds for the modulation of p21 ras expression |
AU2007211080B9 (en) | 2006-01-27 | 2012-05-03 | Isis Pharmaceuticals, Inc. | 6-modified bicyclic nucleic acid analogs |
DK2458006T3 (en) | 2006-05-05 | 2018-08-27 | Ionis Pharmaceuticals Inc | Compounds and Methods for Modulating ApoB Expression. |
WO2007134181A2 (en) | 2006-05-11 | 2007-11-22 | Isis Pharmaceuticals, Inc. | 5'-modified bicyclic nucleic acid analogs |
US7666854B2 (en) | 2006-05-11 | 2010-02-23 | Isis Pharmaceuticals, Inc. | Bis-modified bicyclic nucleic acid analogs |
AU2007339897B2 (en) | 2006-12-27 | 2013-02-14 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for the treatment of infections and tumors |
WO2008113832A2 (en) | 2007-03-22 | 2008-09-25 | Santaris Pharma A/S | SHORT RNA ANTAGONIST COMPOUNDS FOR THE MODULATION OF TARGET mRNA |
EP2170917B1 (en) | 2007-05-30 | 2012-06-27 | Isis Pharmaceuticals, Inc. | N-substituted-aminomethylene bridged bicyclic nucleic acid analogs |
US8278426B2 (en) | 2007-06-08 | 2012-10-02 | Isis Pharmaceuticals, Inc. | Carbocyclic bicyclic nucleic acid analogs |
ATE538127T1 (de) | 2007-07-05 | 2012-01-15 | Isis Pharmaceuticals Inc | 6-disubstituierte bicyclische nukleinsäureanaloga |
US8546556B2 (en) | 2007-11-21 | 2013-10-01 | Isis Pharmaceuticals, Inc | Carbocyclic alpha-L-bicyclic nucleic acid analogs |
WO2009090182A1 (en) | 2008-01-14 | 2009-07-23 | Santaris Pharma A/S | C4'-substituted - dna nucleotide gapmer oligonucleotides |
WO2009124238A1 (en) | 2008-04-04 | 2009-10-08 | Isis Pharmaceuticals, Inc. | Oligomeric compounds comprising neutrally linked terminal bicyclic nucleosides |
WO2009126933A2 (en) | 2008-04-11 | 2009-10-15 | Alnylam Pharmaceuticals, Inc. | Site-specific delivery of nucleic acids by combining targeting ligands with endosomolytic components |
EP2356129B1 (en) | 2008-09-24 | 2013-04-03 | Isis Pharmaceuticals, Inc. | Substituted alpha-l-bicyclic nucleosides |
US9598491B2 (en) | 2008-11-28 | 2017-03-21 | Emory University | Methods for the treatment of infections and tumors |
PE20120341A1 (es) * | 2008-12-09 | 2012-04-24 | Genentech Inc | Anticuerpos anti-pd-l1 y su uso para mejorar la funcion de celulas t |
US9181525B2 (en) * | 2009-03-06 | 2015-11-10 | Mie University | Method for enhancing a function of a T cell |
US9012421B2 (en) | 2009-08-06 | 2015-04-21 | Isis Pharmaceuticals, Inc. | Bicyclic cyclohexose nucleic acid analogs |
US8507663B2 (en) * | 2010-04-06 | 2013-08-13 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of CD274/PD-L1 gene |
CN103370414A (zh) * | 2010-05-18 | 2013-10-23 | 皇家学习促进学会/麦吉尔大学 | 降低恶性神经胶质瘤中下调肾细胞癌的表达的方法 |
WO2011156202A1 (en) | 2010-06-08 | 2011-12-15 | Isis Pharmaceuticals, Inc. | Substituted 2 '-amino and 2 '-thio-bicyclic nucleosides and oligomeric compounds prepared therefrom |
HUE044815T2 (hu) | 2010-08-17 | 2019-11-28 | Sirna Therapeutics Inc | Hepatitisz B vírus (HBV) génexpressziójának RNS-interferencia közvetített gátlása, rövid interferáló nukleinsav (SINS) alkalmazásával |
KR101903778B1 (ko) | 2010-10-28 | 2018-10-04 | 베니텍 바이오파마 리미티드 | Hbv 치료 |
AU2011343664B2 (en) | 2010-12-17 | 2015-10-08 | Arrowhead Pharmaceuticals, Inc. | Galactose cluster-pharmacokinetic modulator targeting moiety for siRNA |
CN104328121A (zh) | 2010-12-29 | 2015-02-04 | 弗·哈夫曼-拉罗切有限公司 | 用于细胞内递送核酸的小分子缀合物 |
CR20190207A (es) | 2011-04-21 | 2019-06-26 | Ionis Pharmaceuticals Inc | MODULACIÓN DE LA EXPRESIÓN DEL VIRUS DE LA HEPATITIS B (VHB) (Divisional 2013-0551) |
KR102434357B1 (ko) | 2011-06-30 | 2022-08-18 | 애로우헤드 파마슈티컬스 인코포레이티드 | B형 간염 바이러스의 유전자 발현 저해용 조성물 및 방법 |
JP6128529B2 (ja) | 2011-07-19 | 2017-05-17 | ウェイブ ライフ サイエンシズ リミテッドWave Life Sciences Ltd. | 官能化核酸の合成のための方法 |
WO2013033230A1 (en) | 2011-08-29 | 2013-03-07 | Isis Pharmaceuticals, Inc. | Oligomer-conjugate complexes and their use |
CA2863253A1 (en) | 2011-09-07 | 2013-03-14 | Marina Biotech, Inc. | Synthesis and uses of nucleic acid compounds with conformationally restricted monomers |
WO2013049307A2 (en) | 2011-09-30 | 2013-04-04 | University Of Miami | Enhanced immune memory development by aptamer targeted mtor inhibition of t cells |
DK2768524T3 (da) * | 2011-10-17 | 2022-07-04 | Io Biotech Aps | PD-L1-baseret immunterapi |
US9221864B2 (en) | 2012-04-09 | 2015-12-29 | Isis Pharmaceuticals, Inc. | Tricyclic nucleic acid analogs |
WO2013159109A1 (en) | 2012-04-20 | 2013-10-24 | Isis Pharmaceuticals, Inc. | Modulation of hepatitis b virus (hbv) expression |
US20150232836A1 (en) | 2012-05-16 | 2015-08-20 | Rana Therapeutics, Inc. | Compositions and methods for modulating gene expression |
CN104583401A (zh) * | 2012-05-16 | 2015-04-29 | Rana医疗有限公司 | 用于调节atp2a2表达的组合物和方法 |
EP2872485B1 (en) | 2012-07-13 | 2020-12-16 | Wave Life Sciences Ltd. | Asymmetric auxiliary group |
RU2653438C2 (ru) | 2012-11-15 | 2018-05-08 | Рош Инновейшен Сентер Копенгаген А/С | Конъюгаты олигонуклеотидов |
EP2926142B2 (en) | 2012-11-30 | 2022-07-06 | F. Hoffmann-La Roche AG | Identification of patients in need of pd-l1 inhibitor cotherapy |
WO2014118272A1 (en) | 2013-01-30 | 2014-08-07 | Santaris Pharma A/S | Antimir-122 oligonucleotide carbohydrate conjugates |
PE20152002A1 (es) | 2013-05-01 | 2016-01-21 | Isis Pharmaceuticals Inc | Composiciones y metodos para modular la expresion de ttr y vhb |
LT3013959T (lt) | 2013-06-27 | 2020-03-10 | Roche Innovation Center Copenhagen A/S | Priešprasmiai oligomerai ir konjugatai, nukreirti į pcsk9 |
EP3102697A1 (en) * | 2014-02-03 | 2016-12-14 | Myriad Genetics, Inc. | Method for predicting the response to an anti-her2 containing therapy and/or chemotherapy in patients with breast cancer |
WO2016025645A1 (en) | 2014-08-12 | 2016-02-18 | Massachusetts Institute Of Technology | Synergistic tumor treatment with il-2, a therapeutic antibody, and an immune checkpoint blocker |
KR20170068469A (ko) | 2014-10-10 | 2017-06-19 | 에프. 호프만-라 로슈 아게 | GalNAc 포스포라미다이트, 그의 핵산 컨쥬게이트 및 그의 용도 |
WO2016138278A2 (en) | 2015-02-27 | 2016-09-01 | Idera Pharmaceuticals, Inc. | Compositions for inhibiting checkpoint gene expression and uses thereof |
GB201507926D0 (en) | 2015-05-08 | 2015-06-24 | Proqr Therapeutics N V | Improved treatments using oligonucleotides |
WO2017055423A1 (en) * | 2015-10-02 | 2017-04-06 | Roche Innovation Center Copenhagen A/S | Oligonucleotide conjugation process |
WO2017084987A1 (en) | 2015-11-16 | 2017-05-26 | F. Hoffmann-La Roche Ag | GalNAc CLUSTER PHOSPHORAMIDITE |
WO2017100587A1 (en) | 2015-12-09 | 2017-06-15 | Alnylam Pharmaceuticals, Inc. | Polynucleotide agents targeting programmed cell death 1 ligand 1 (pd-l1) and methods of use thereof |
DK3430141T3 (da) | 2016-03-14 | 2021-03-01 | Hoffmann La Roche | Oligonukleotider til reduktion af PD-L1-ekspression |
-
2017
- 2017-03-14 DK DK17710874.3T patent/DK3430141T3/da active
- 2017-03-14 BR BR112018068410A patent/BR112018068410A2/pt active Search and Examination
- 2017-03-14 PE PE2018001567A patent/PE20181892A1/es unknown
- 2017-03-14 RS RS20210240A patent/RS61528B1/sr unknown
- 2017-03-14 ES ES17710874T patent/ES2857702T3/es active Active
- 2017-03-14 KR KR1020227022628A patent/KR102580776B1/ko active IP Right Grant
- 2017-03-14 TW TW106108305A patent/TWI721128B/zh active
- 2017-03-14 CN CN201780017220.7A patent/CN108779465B/zh active Active
- 2017-03-14 KR KR1020217030513A patent/KR102417646B1/ko active IP Right Grant
- 2017-03-14 CR CR20200119A patent/CR20200119A/es unknown
- 2017-03-14 EP EP20201780.2A patent/EP3786297A1/en active Pending
- 2017-03-14 CA CA3120687A patent/CA3120687A1/en active Pending
- 2017-03-14 IL IL296483A patent/IL296483B2/en unknown
- 2017-03-14 KR KR1020237031671A patent/KR20230136689A/ko not_active Application Discontinuation
- 2017-03-14 JP JP2018548393A patent/JP6748219B2/ja active Active
- 2017-03-14 MY MYPI2018703228A patent/MY194912A/en unknown
- 2017-03-14 RU RU2018134379A patent/RU2747822C2/ru active
- 2017-03-14 TW TW109135754A patent/TWI794662B/zh active
- 2017-03-14 LT LTEP17710874.3T patent/LT3430141T/lt unknown
- 2017-03-14 AU AU2017235278A patent/AU2017235278C1/en active Active
- 2017-03-14 MX MX2018010830A patent/MX2018010830A/es unknown
- 2017-03-14 EP EP17710874.3A patent/EP3430141B1/en active Active
- 2017-03-14 PE PE2022000852A patent/PE20230157A1/es unknown
- 2017-03-14 HU HUE17710874A patent/HUE053172T2/hu unknown
- 2017-03-14 PT PT177108743T patent/PT3430141T/pt unknown
- 2017-03-14 AR ARP170100626A patent/AR108038A1/es unknown
- 2017-03-14 CN CN202210440723.8A patent/CN114717235A/zh active Pending
- 2017-03-14 SG SG11201807854SA patent/SG11201807854SA/en unknown
- 2017-03-14 IL IL303077A patent/IL303077A/en unknown
- 2017-03-14 PE PE2020000807A patent/PE20201499A1/es unknown
- 2017-03-14 CN CN202210442730.1A patent/CN114736900B/zh active Active
- 2017-03-14 TW TW109135755A patent/TWI790485B/zh active
- 2017-03-14 WO PCT/EP2017/055925 patent/WO2017157899A1/en active Application Filing
- 2017-03-14 PL PL17710874T patent/PL3430141T3/pl unknown
- 2017-03-14 US US15/458,800 patent/US20170283496A1/en not_active Abandoned
- 2017-03-14 UA UAA201810179A patent/UA127432C2/uk unknown
- 2017-03-14 CN CN202111366505.6A patent/CN114085836B/zh active Active
- 2017-03-14 SI SI201730645T patent/SI3430141T1/sl unknown
- 2017-03-14 KR KR1020187026546A patent/KR102306797B1/ko active IP Right Grant
- 2017-03-14 CA CA3013683A patent/CA3013683C/en active Active
- 2017-03-14 CR CR20180432A patent/CR20180432A/es unknown
-
2018
- 2018-07-24 IL IL260759A patent/IL260759B/en unknown
- 2018-07-26 CO CONC2018/0007761A patent/CO2018007761A2/es unknown
- 2018-09-07 CL CL2018002570A patent/CL2018002570A1/es unknown
- 2018-09-07 MX MX2022012221A patent/MX2022012221A/es unknown
- 2018-09-12 PH PH12018501964A patent/PH12018501964A1/en unknown
-
2019
- 2019-10-25 US US16/664,749 patent/US10745480B2/en active Active
-
2020
- 2020-03-31 CL CL2020000865A patent/CL2020000865A1/es unknown
- 2020-04-02 US US16/839,025 patent/US10829555B2/en active Active
- 2020-04-17 AR ARP200101090A patent/AR118719A2/es unknown
- 2020-04-28 CL CL2020001126A patent/CL2020001126A1/es unknown
- 2020-04-28 CL CL2020001127A patent/CL2020001127A1/es unknown
- 2020-06-22 JP JP2020106728A patent/JP7002603B2/ja active Active
- 2020-08-21 US US17/000,203 patent/US11466081B2/en active Active
-
2021
- 2021-02-25 HR HRP20210315TT patent/HRP20210315T1/hr unknown
- 2021-09-20 AU AU2021236439A patent/AU2021236439B2/en active Active
- 2021-12-27 JP JP2021212541A patent/JP7447073B2/ja active Active
-
2022
- 2022-02-01 IL IL290294A patent/IL290294B2/en unknown
- 2022-04-13 AU AU2022202479A patent/AU2022202479A1/en active Pending
- 2022-10-07 US US18/045,109 patent/US20230331837A1/en active Pending
-
2024
- 2024-01-04 JP JP2024000251A patent/JP2024029180A/ja active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010129799A2 (en) * | 2009-05-06 | 2010-11-11 | Curna, Inc. | Treatment of lipid transport and metabolism gene related diseases by inhibition of natural antisense transcript to a lipid transport and metabolism gene |
US20140288153A1 (en) * | 2009-05-28 | 2014-09-25 | Curna, Inc. | Treatment of antiviral gene related diseases by inhibition of natural antisense transcript to an antiviral gene |
WO2014118267A1 (en) * | 2013-01-30 | 2014-08-07 | Santaris Pharma A/S | Lna oligonucleotide carbohydrate conjugates |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10745480B2 (en) | 2016-03-14 | 2020-08-18 | Hoffmann-La Roche, Inc. | Oligonucleotides for reduction of PD-L1 expression |
US10829555B2 (en) | 2016-03-14 | 2020-11-10 | Hoffman-La Roche Inc. | Oligonucleotides for reduction of PD-L1 expression |
US11466081B2 (en) | 2016-03-14 | 2022-10-11 | Hoffmann-La Roche Inc. | Oligonucleotides for reduction of PD-L1 expression |
CN111511914A (zh) * | 2017-10-16 | 2020-08-07 | 豪夫迈·罗氏有限公司 | 减少PAPD5和PAPD7 mRNA的核酸分子用于治疗乙型肝炎感染 |
CN111819283A (zh) * | 2018-01-12 | 2020-10-23 | 百时美施贵宝公司 | 靶向α-突触核蛋白的反义寡核苷酸及其用途 |
WO2020081585A1 (en) * | 2018-10-15 | 2020-04-23 | The Brigham And Women's Hospital, Inc. | The long non-coding rna inca1 and homo sapiens heterogeneous nuclear ribonucleoprotein h1 (hnrnph1) as therapeutic targets for immunotherapy |
US20210214727A1 (en) * | 2019-12-20 | 2021-07-15 | Hoffmann-La Roche Inc. | Enhanced oligonucleotides for inhibiting scn9a expression |
WO2024011109A3 (en) * | 2022-07-06 | 2024-04-11 | Adverum Biotechnologies, Inc. | Compositions and methods for treatment of achromotopsia |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11466081B2 (en) | Oligonucleotides for reduction of PD-L1 expression | |
NZ785335A (en) | Oligonucleotides for reduction of pd-l1 expression | |
NZ785334A (en) | Oligonucleotides for reduction of pd-l1 expression | |
TWI840950B (zh) | 用於降低pd-l1表現之寡核苷酸 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ROCHE INNOVATION CENTER COPENHAGEN A/S, DENMARK Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:PEDERSEN, LYKKE;OTTOSEN, SOREN;JACKEROTT, MALENE;SIGNING DATES FROM 20160627 TO 20160822;REEL/FRAME:043009/0576 Owner name: HOFFMANN-LA ROCHE INC., NEW JERSEY Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:F. HOFFMANN-LA ROCHE AG;REEL/FRAME:043010/0224 Effective date: 20170313 Owner name: F. HOFFMANN-LA ROCHE AG, SWITZERLAND Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:HASSAN JAVANBAKHT;REEL/FRAME:043009/0845 Effective date: 20160524 Owner name: F. HOFFMANN-LA ROCHE AG, SWITZERLAND Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SOUPHALONE LUANGSAY;REEL/FRAME:043010/0046 Effective date: 20170301 Owner name: F. HOFFMANN-LA ROCHE AG, SWITZERLAND Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ROCHE INNOVATION CENTER COPENHAGEN A/S;REEL/FRAME:043010/0157 Effective date: 20160907 |
|
AS | Assignment |
Owner name: HOFFMANN-LA ROCHE INC., NEW JERSEY Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ROCHE INNOVATION CENTER COPENHAGEN A/S;REEL/FRAME:044768/0961 Effective date: 20171211 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NOTICE OF ALLOWANCE MAILED -- APPLICATION RECEIVED IN OFFICE OF PUBLICATIONS |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO PAY ISSUE FEE |