EP1660658A2 - The use of sirna silencing in the prevention of metastasis - Google Patents
The use of sirna silencing in the prevention of metastasisInfo
- Publication number
- EP1660658A2 EP1660658A2 EP04816701A EP04816701A EP1660658A2 EP 1660658 A2 EP1660658 A2 EP 1660658A2 EP 04816701 A EP04816701 A EP 04816701A EP 04816701 A EP04816701 A EP 04816701A EP 1660658 A2 EP1660658 A2 EP 1660658A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- sirna
- seq
- use according
- cells
- sirnas
- 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.)
- Withdrawn
Links
- 206010027476 Metastases Diseases 0.000 title claims abstract description 48
- 230000009401 metastasis Effects 0.000 title claims abstract description 47
- 230000002265 prevention Effects 0.000 title claims abstract description 12
- 230000006807 siRNA silencing Effects 0.000 title description 6
- 108020004459 Small interfering RNA Proteins 0.000 claims abstract description 318
- 108010000499 Thromboplastin Proteins 0.000 claims abstract description 174
- 102000002262 Thromboplastin Human genes 0.000 claims abstract description 173
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 76
- 108020004999 messenger RNA Proteins 0.000 claims description 66
- 239000002773 nucleotide Substances 0.000 claims description 43
- 125000003729 nucleotide group Chemical group 0.000 claims description 39
- 239000000203 mixture Substances 0.000 claims description 37
- 150000007523 nucleic acids Chemical group 0.000 claims description 28
- 239000004055 small Interfering RNA Substances 0.000 claims description 23
- 241000282414 Homo sapiens Species 0.000 claims description 20
- 230000000295 complement effect Effects 0.000 claims description 18
- 238000003776 cleavage reaction Methods 0.000 claims description 15
- 230000007017 scission Effects 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 11
- 238000009472 formulation Methods 0.000 claims description 11
- 238000001990 intravenous administration Methods 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 7
- 239000008194 pharmaceutical composition Substances 0.000 claims description 7
- 239000002671 adjuvant Substances 0.000 claims description 6
- 239000000443 aerosol Substances 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 6
- 238000001802 infusion Methods 0.000 claims description 6
- 238000007920 subcutaneous administration Methods 0.000 claims description 6
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims description 5
- 238000010521 absorption reaction Methods 0.000 claims description 5
- 238000007918 intramuscular administration Methods 0.000 claims description 5
- 239000007927 intramuscular injection Substances 0.000 claims description 5
- 239000007928 intraperitoneal injection Substances 0.000 claims description 5
- 239000000829 suppository Substances 0.000 claims description 5
- 239000002775 capsule Substances 0.000 claims description 4
- 239000000969 carrier Substances 0.000 claims description 4
- 239000003085 diluting agent Substances 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- 239000003826 tablet Substances 0.000 claims description 4
- 239000011230 binding agent Substances 0.000 claims description 3
- 238000004040 coloring Methods 0.000 claims description 3
- 239000000796 flavoring agent Substances 0.000 claims description 3
- 235000003599 food sweetener Nutrition 0.000 claims description 3
- 239000000314 lubricant Substances 0.000 claims description 3
- 231100000252 nontoxic Toxicity 0.000 claims description 3
- 230000003000 nontoxic effect Effects 0.000 claims description 3
- 239000006187 pill Substances 0.000 claims description 3
- 238000013268 sustained release Methods 0.000 claims description 3
- 239000012730 sustained-release form Substances 0.000 claims description 3
- 239000003765 sweetening agent Substances 0.000 claims description 3
- 239000003978 infusion fluid Substances 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 abstract description 65
- 230000014509 gene expression Effects 0.000 abstract description 62
- 102000040650 (ribonucleotides)n+m Human genes 0.000 abstract description 39
- 238000011282 treatment Methods 0.000 abstract description 20
- 201000011510 cancer Diseases 0.000 abstract description 15
- 210000004027 cell Anatomy 0.000 description 114
- 230000000694 effects Effects 0.000 description 75
- 230000009368 gene silencing by RNA Effects 0.000 description 44
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 41
- 238000000034 method Methods 0.000 description 37
- 108091034117 Oligonucleotide Proteins 0.000 description 31
- 108090000623 proteins and genes Proteins 0.000 description 30
- 230000035772 mutation Effects 0.000 description 27
- 238000001890 transfection Methods 0.000 description 27
- 238000003556 assay Methods 0.000 description 26
- 239000007924 injection Substances 0.000 description 26
- 238000002347 injection Methods 0.000 description 26
- 241000699670 Mus sp. Species 0.000 description 25
- 238000002474 experimental method Methods 0.000 description 24
- 150000003839 salts Chemical class 0.000 description 24
- 230000030279 gene silencing Effects 0.000 description 22
- 238000012986 modification Methods 0.000 description 21
- 101000635804 Homo sapiens Tissue factor Proteins 0.000 description 20
- 108091027967 Small hairpin RNA Proteins 0.000 description 20
- 230000004048 modification Effects 0.000 description 20
- -1 genomic Proteins 0.000 description 17
- 102000039446 nucleic acids Human genes 0.000 description 17
- 108020004707 nucleic acids Proteins 0.000 description 17
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 16
- 238000004458 analytical method Methods 0.000 description 16
- 108010002380 serum tumorlytic factor Proteins 0.000 description 16
- 239000002253 acid Substances 0.000 description 15
- 230000001404 mediated effect Effects 0.000 description 15
- 206010053567 Coagulopathies Diseases 0.000 description 13
- 239000002585 base Substances 0.000 description 13
- 150000001875 compounds Chemical class 0.000 description 13
- 230000008685 targeting Effects 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- 108060001084 Luciferase Proteins 0.000 description 12
- 239000005089 Luciferase Substances 0.000 description 12
- 238000006731 degradation reaction Methods 0.000 description 12
- 210000004962 mammalian cell Anatomy 0.000 description 12
- 241000124008 Mammalia Species 0.000 description 11
- 230000000692 anti-sense effect Effects 0.000 description 11
- 230000015556 catabolic process Effects 0.000 description 11
- 230000001225 therapeutic effect Effects 0.000 description 11
- 238000000338 in vitro Methods 0.000 description 10
- 210000004072 lung Anatomy 0.000 description 10
- 230000002829 reductive effect Effects 0.000 description 10
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 9
- 239000002777 nucleoside Substances 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 241000894007 species Species 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 102000000574 RNA-Induced Silencing Complex Human genes 0.000 description 8
- 108010016790 RNA-Induced Silencing Complex Proteins 0.000 description 8
- 125000000217 alkyl group Chemical group 0.000 description 8
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 230000001965 increasing effect Effects 0.000 description 8
- 230000001394 metastastic effect Effects 0.000 description 8
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 108090000994 Catalytic RNA Proteins 0.000 description 7
- 102000053642 Catalytic RNA Human genes 0.000 description 7
- 208000001382 Experimental Melanoma Diseases 0.000 description 7
- 102000006382 Ribonucleases Human genes 0.000 description 7
- 108010083644 Ribonucleases Proteins 0.000 description 7
- 230000033115 angiogenesis Effects 0.000 description 7
- 230000035602 clotting Effects 0.000 description 7
- 239000002299 complementary DNA Substances 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 7
- 230000003211 malignant effect Effects 0.000 description 7
- 206010061289 metastatic neoplasm Diseases 0.000 description 7
- 230000037361 pathway Effects 0.000 description 7
- 108091092562 ribozyme Proteins 0.000 description 7
- 230000009885 systemic effect Effects 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 6
- 108091081021 Sense strand Proteins 0.000 description 6
- 238000007792 addition Methods 0.000 description 6
- 230000004071 biological effect Effects 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 208000015294 blood coagulation disease Diseases 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 230000009852 coagulant defect Effects 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 238000003197 gene knockdown Methods 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 230000000977 initiatory effect Effects 0.000 description 6
- 230000007246 mechanism Effects 0.000 description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 6
- 125000003835 nucleoside group Chemical group 0.000 description 6
- 230000002947 procoagulating effect Effects 0.000 description 6
- 230000002685 pulmonary effect Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 241000283690 Bos taurus Species 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000012226 gene silencing method Methods 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 239000002502 liposome Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 230000002688 persistence Effects 0.000 description 5
- 229910052698 phosphorus Inorganic materials 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 239000013600 plasmid vector Substances 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 4
- 108091093037 Peptide nucleic acid Proteins 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 4
- 150000001412 amines Chemical class 0.000 description 4
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 4
- 230000023555 blood coagulation Effects 0.000 description 4
- 210000004556 brain Anatomy 0.000 description 4
- 150000001768 cations Chemical class 0.000 description 4
- 229940104302 cytosine Drugs 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 230000009977 dual effect Effects 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 238000003306 harvesting Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000007912 intraperitoneal administration Methods 0.000 description 4
- 125000005647 linker group Chemical group 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 210000001161 mammalian embryo Anatomy 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 4
- 150000003833 nucleoside derivatives Chemical class 0.000 description 4
- 239000000546 pharmaceutical excipient Substances 0.000 description 4
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 4
- 125000004437 phosphorous atom Chemical group 0.000 description 4
- 230000026731 phosphorylation Effects 0.000 description 4
- 238000006366 phosphorylation reaction Methods 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 230000001603 reducing effect Effects 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 4
- 229930024421 Adenine Natural products 0.000 description 3
- 108020005544 Antisense RNA Proteins 0.000 description 3
- 239000012983 Dulbecco’s minimal essential medium Substances 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 108090001102 Hammerhead ribozyme Proteins 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Natural products O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 3
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 3
- 150000007513 acids Chemical class 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 229960000643 adenine Drugs 0.000 description 3
- 125000003342 alkenyl group Chemical group 0.000 description 3
- 230000004075 alteration Effects 0.000 description 3
- 150000001408 amides Chemical group 0.000 description 3
- 210000003484 anatomy Anatomy 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000007385 chemical modification Methods 0.000 description 3
- 238000007820 coagulation assay Methods 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 230000001747 exhibiting effect Effects 0.000 description 3
- 230000037440 gene silencing effect Effects 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 125000000623 heterocyclic group Chemical group 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 238000010253 intravenous injection Methods 0.000 description 3
- 208000037841 lung tumor Diseases 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 150000007522 mineralic acids Chemical class 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 150000007524 organic acids Chemical class 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 229940002612 prodrug Drugs 0.000 description 3
- 239000000651 prodrug Substances 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 125000002652 ribonucleotide group Chemical group 0.000 description 3
- 125000001424 substituent group Chemical group 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 3
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 230000001052 transient effect Effects 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- 229940035893 uracil Drugs 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 2
- FZWGECJQACGGTI-UHFFFAOYSA-N 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one Chemical compound NC1=NC(O)=C2N(C)C=NC2=N1 FZWGECJQACGGTI-UHFFFAOYSA-N 0.000 description 2
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 2
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 2
- RYVNIFSIEDRLSJ-UHFFFAOYSA-N 5-(hydroxymethyl)cytosine Chemical compound NC=1NC(=O)N=CC=1CO RYVNIFSIEDRLSJ-UHFFFAOYSA-N 0.000 description 2
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 2
- 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 2
- PEHVGBZKEYRQSX-UHFFFAOYSA-N 7-deaza-adenine Chemical compound NC1=NC=NC2=C1C=CN2 PEHVGBZKEYRQSX-UHFFFAOYSA-N 0.000 description 2
- HCGHYQLFMPXSDU-UHFFFAOYSA-N 7-methyladenine Chemical compound C1=NC(N)=C2N(C)C=NC2=N1 HCGHYQLFMPXSDU-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- 239000005711 Benzoic acid Substances 0.000 description 2
- 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 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 102100023804 Coagulation factor VII Human genes 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 2
- 241000255601 Drosophila melanogaster Species 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 108010023321 Factor VII Proteins 0.000 description 2
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 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 2
- 102100034343 Integrase Human genes 0.000 description 2
- 101710203526 Integrase Proteins 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- 206010027458 Metastases to lung Diseases 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 241000244206 Nematoda Species 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 238000000636 Northern blotting Methods 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 108010009341 Protein Serine-Threonine Kinases Proteins 0.000 description 2
- 102000009516 Protein Serine-Threonine Kinases Human genes 0.000 description 2
- 108010094028 Prothrombin Proteins 0.000 description 2
- 102100027378 Prothrombin Human genes 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 241000251131 Sphyrna Species 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 241001150496 Tosapusia duplex Species 0.000 description 2
- 108700019146 Transgenes Proteins 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
- 235000011054 acetic acid Nutrition 0.000 description 2
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 2
- 125000000304 alkynyl group Chemical group 0.000 description 2
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 239000002257 antimetastatic agent Substances 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 235000010233 benzoic acid Nutrition 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
- 229910052794 bromium Inorganic materials 0.000 description 2
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000004700 cellular uptake Effects 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical group 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
- 235000015165 citric acid Nutrition 0.000 description 2
- 230000015271 coagulation Effects 0.000 description 2
- 238000005345 coagulation Methods 0.000 description 2
- 239000003184 complementary RNA Substances 0.000 description 2
- 125000000753 cycloalkyl group Chemical group 0.000 description 2
- HCAJEUSONLESMK-UHFFFAOYSA-N cyclohexylsulfamic acid Chemical compound OS(=O)(=O)NC1CCCCC1 HCAJEUSONLESMK-UHFFFAOYSA-N 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 230000002222 downregulating effect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000003511 endothelial effect Effects 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 239000001530 fumaric acid Substances 0.000 description 2
- 235000011087 fumaric acid Nutrition 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000001476 gene delivery Methods 0.000 description 2
- 239000000174 gluconic acid Substances 0.000 description 2
- 235000012208 gluconic acid Nutrition 0.000 description 2
- 229940093915 gynecological organic acid Drugs 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 125000005842 heteroatom Chemical group 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 239000003999 initiator Substances 0.000 description 2
- 238000001361 intraarterial administration Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000007917 intracranial administration Methods 0.000 description 2
- 238000007913 intrathecal administration Methods 0.000 description 2
- 238000007914 intraventricular administration Methods 0.000 description 2
- 230000009545 invasion Effects 0.000 description 2
- SUMDYPCJJOFFON-UHFFFAOYSA-N isethionic acid Chemical compound OCCS(O)(=O)=O SUMDYPCJJOFFON-UHFFFAOYSA-N 0.000 description 2
- TWBYWOBDOCUKOW-UHFFFAOYSA-N isonicotinic acid Chemical compound OC(=O)C1=CC=NC=C1 TWBYWOBDOCUKOW-UHFFFAOYSA-N 0.000 description 2
- 210000002510 keratinocyte Anatomy 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- JJTUDXZGHPGLLC-UHFFFAOYSA-N lactide Chemical compound CC1OC(=O)C(C)OC1=O JJTUDXZGHPGLLC-UHFFFAOYSA-N 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 238000010841 mRNA extraction Methods 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 2
- 239000011976 maleic acid Substances 0.000 description 2
- 239000001630 malic acid Substances 0.000 description 2
- 235000011090 malic acid Nutrition 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 150000002739 metals Chemical class 0.000 description 2
- 229940098779 methanesulfonic acid Drugs 0.000 description 2
- 230000011987 methylation Effects 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 2
- 239000007923 nasal drop Substances 0.000 description 2
- 229940100662 nasal drops Drugs 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- 235000006408 oxalic acid Nutrition 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 229920000768 polyamine Polymers 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- 229940039716 prothrombin Drugs 0.000 description 2
- 150000003212 purines Chemical class 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 2
- 230000010473 stable expression Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000011477 surgical intervention Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 239000011975 tartaric acid Substances 0.000 description 2
- 235000002906 tartaric acid Nutrition 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- QBYIENPQHBMVBV-HFEGYEGKSA-N (2R)-2-hydroxy-2-phenylacetic acid Chemical compound O[C@@H](C(O)=O)c1ccccc1.O[C@@H](C(O)=O)c1ccccc1 QBYIENPQHBMVBV-HFEGYEGKSA-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
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 description 1
- QGVQZRDQPDLHHV-DPAQBDIFSA-N (3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthrene-3-thiol Chemical compound C1C=C2C[C@@H](S)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 QGVQZRDQPDLHHV-DPAQBDIFSA-N 0.000 description 1
- UWYVPFMHMJIBHE-OWOJBTEDSA-N (e)-2-hydroxybut-2-enedioic acid Chemical compound OC(=O)\C=C(\O)C(O)=O UWYVPFMHMJIBHE-OWOJBTEDSA-N 0.000 description 1
- HCUOEKSZWPGJIM-YBRHCDHNSA-N (e,2e)-2-hydroxyimino-6-methoxy-4-methyl-5-nitrohex-3-enamide Chemical compound COCC([N+]([O-])=O)\C(C)=C\C(=N/O)\C(N)=O HCUOEKSZWPGJIM-YBRHCDHNSA-N 0.000 description 1
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 1
- FYADHXFMURLYQI-UHFFFAOYSA-N 1,2,4-triazine Chemical class C1=CN=NC=N1 FYADHXFMURLYQI-UHFFFAOYSA-N 0.000 description 1
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- UHUHBFMZVCOEOV-UHFFFAOYSA-N 1h-imidazo[4,5-c]pyridin-4-amine Chemical compound NC1=NC=CC2=C1N=CN2 UHUHBFMZVCOEOV-UHFFFAOYSA-N 0.000 description 1
- QSHACTSJHMKXTE-UHFFFAOYSA-N 2-(2-aminopropyl)-7h-purin-6-amine Chemical compound CC(N)CC1=NC(N)=C2NC=NC2=N1 QSHACTSJHMKXTE-UHFFFAOYSA-N 0.000 description 1
- PIINGYXNCHTJTF-UHFFFAOYSA-N 2-(2-azaniumylethylamino)acetate Chemical group NCCNCC(O)=O PIINGYXNCHTJTF-UHFFFAOYSA-N 0.000 description 1
- 125000004200 2-methoxyethyl group Chemical group [H]C([H])([H])OC([H])([H])C([H])([H])* 0.000 description 1
- PKRSYEPBQPFNRB-UHFFFAOYSA-N 2-phenoxybenzoic acid Chemical compound OC(=O)C1=CC=CC=C1OC1=CC=CC=C1 PKRSYEPBQPFNRB-UHFFFAOYSA-N 0.000 description 1
- WLJVXDMOQOGPHL-PPJXEINESA-N 2-phenylacetic acid Chemical compound O[14C](=O)CC1=CC=CC=C1 WLJVXDMOQOGPHL-PPJXEINESA-N 0.000 description 1
- GXIURPTVHJPJLF-UHFFFAOYSA-N 2-phosphoglyceric acid Chemical compound OCC(C(O)=O)OP(O)(O)=O GXIURPTVHJPJLF-UHFFFAOYSA-N 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- WUBBRNOQWQTFEX-UHFFFAOYSA-N 4-aminosalicylic acid Chemical compound NC1=CC=C(C(O)=O)C(O)=C1 WUBBRNOQWQTFEX-UHFFFAOYSA-N 0.000 description 1
- OVONXEQGWXGFJD-UHFFFAOYSA-N 4-sulfanylidene-1h-pyrimidin-2-one Chemical compound SC=1C=CNC(=O)N=1 OVONXEQGWXGFJD-UHFFFAOYSA-N 0.000 description 1
- IJJWOSAXNHWBPR-HUBLWGQQSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-n-(6-hydrazinyl-6-oxohexyl)pentanamide Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)NCCCCCC(=O)NN)SC[C@@H]21 IJJWOSAXNHWBPR-HUBLWGQQSA-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
- KXBCLNRMQPRVTP-UHFFFAOYSA-N 6-amino-1,5-dihydroimidazo[4,5-c]pyridin-4-one Chemical compound O=C1NC(N)=CC2=C1N=CN2 KXBCLNRMQPRVTP-UHFFFAOYSA-N 0.000 description 1
- DCPSTSVLRXOYGS-UHFFFAOYSA-N 6-amino-1h-pyrimidine-2-thione Chemical compound NC1=CC=NC(S)=N1 DCPSTSVLRXOYGS-UHFFFAOYSA-N 0.000 description 1
- 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
- LOSIULRWFAEMFL-UHFFFAOYSA-N 7-deazaguanine Chemical compound O=C1NC(N)=NC2=C1CC=N2 LOSIULRWFAEMFL-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- HRYKDUPGBWLLHO-UHFFFAOYSA-N 8-azaadenine Chemical compound NC1=NC=NC2=NNN=C12 HRYKDUPGBWLLHO-UHFFFAOYSA-N 0.000 description 1
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 1
- 229960005508 8-azaguanine Drugs 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
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010083590 Apoproteins Proteins 0.000 description 1
- 102000006410 Apoproteins Human genes 0.000 description 1
- 206010003178 Arterial thrombosis Diseases 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 208000037260 Atherosclerotic Plaque Diseases 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 239000004380 Cholic acid Substances 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 1
- 101100007328 Cocos nucifera COS-1 gene Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 1
- DSLZVSRJTYRBFB-LLEIAEIESA-N D-glucaric acid Chemical compound OC(=O)[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O DSLZVSRJTYRBFB-LLEIAEIESA-N 0.000 description 1
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 1
- 230000007067 DNA methylation Effects 0.000 description 1
- XBPCUCUWBYBCDP-UHFFFAOYSA-N Dicyclohexylamine Chemical compound C1CCCCC1NC1CCCCC1 XBPCUCUWBYBCDP-UHFFFAOYSA-N 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000991587 Enterovirus C Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 108010007979 Glycocholic Acid Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001006996 Homo sapiens Serine/threonine-protein kinase H1 Proteins 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
- 235000003332 Ilex aquifolium Nutrition 0.000 description 1
- 241000209027 Ilex aquifolium Species 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-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
- 101710141452 Major surface glycoprotein G Proteins 0.000 description 1
- 241001599018 Melanogaster Species 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- MBBZMMPHUWSWHV-BDVNFPICSA-N N-methylglucamine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO MBBZMMPHUWSWHV-BDVNFPICSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- JCXJVPUVTGWSNB-UHFFFAOYSA-N Nitrogen dioxide Chemical compound O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 229910004679 ONO2 Inorganic materials 0.000 description 1
- REYJJPSVUYRZGE-UHFFFAOYSA-N Octadecylamine Chemical compound CCCCCCCCCCCCCCCCCCN REYJJPSVUYRZGE-UHFFFAOYSA-N 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 241001504519 Papio ursinus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229920002230 Pectic acid Polymers 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- ABLZXFCXXLZCGV-UHFFFAOYSA-N Phosphorous acid Chemical class OP(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108010020346 Polyglutamic Acid Proteins 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- IWYDHOAUDWTVEP-UHFFFAOYSA-N R-2-phenyl-2-hydroxyacetic acid Natural products OC(=O)C(O)C1=CC=CC=C1 IWYDHOAUDWTVEP-UHFFFAOYSA-N 0.000 description 1
- 102000014450 RNA Polymerase III Human genes 0.000 description 1
- 108010078067 RNA Polymerase III Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 102100028474 Serine/threonine-protein kinase H1 Human genes 0.000 description 1
- 102000039471 Small Nuclear RNA Human genes 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 102100030951 Tissue factor pathway inhibitor Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- RLXCFCYWFYXTON-JTTSDREOSA-N [(3S,8S,9S,10R,13S,14S,17R)-3-hydroxy-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-16-yl] N-hexylcarbamate Chemical group C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC(OC(=O)NCCCCCC)[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 RLXCFCYWFYXTON-JTTSDREOSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- XVIYCJDWYLJQBG-UHFFFAOYSA-N acetic acid;adamantane Chemical compound CC(O)=O.C1C(C2)CC3CC1CC2C3 XVIYCJDWYLJQBG-UHFFFAOYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001447 alkali salts Chemical class 0.000 description 1
- 150000001342 alkaline earth metals Chemical class 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 125000002877 alkyl aryl group Chemical group 0.000 description 1
- 125000005600 alkyl phosphonate group Chemical group 0.000 description 1
- 238000005937 allylation reaction Methods 0.000 description 1
- 150000001371 alpha-amino acids Chemical class 0.000 description 1
- 235000008206 alpha-amino acids Nutrition 0.000 description 1
- 125000005122 aminoalkylamino group Chemical group 0.000 description 1
- 229960004909 aminosalicylic acid Drugs 0.000 description 1
- MDFFNEOEWAXZRQ-UHFFFAOYSA-N aminyl Chemical compound [NH2] MDFFNEOEWAXZRQ-UHFFFAOYSA-N 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 239000002787 antisense oligonuctleotide Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 229940121357 antivirals Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000003710 aryl alkyl group Chemical group 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 229960002319 barbital Drugs 0.000 description 1
- 230000037429 base substitution Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- JUHORIMYRDESRB-UHFFFAOYSA-N benzathine Chemical compound C=1C=CC=CC=1CNCCNCC1=CC=CC=C1 JUHORIMYRDESRB-UHFFFAOYSA-N 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 description 1
- 229940092714 benzenesulfonic acid Drugs 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 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
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 101150055766 cat gene Proteins 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- VDANGULDQQJODZ-UHFFFAOYSA-N chloroprocaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1Cl VDANGULDQQJODZ-UHFFFAOYSA-N 0.000 description 1
- 229960002023 chloroprocaine Drugs 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 1
- 235000019416 cholic acid Nutrition 0.000 description 1
- 229960002471 cholic acid Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 235000013985 cinnamic acid Nutrition 0.000 description 1
- 229930016911 cinnamic acid Natural products 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- HNEGQIOMVPPMNR-IHWYPQMZSA-N citraconic acid Chemical compound OC(=O)C(/C)=C\C(O)=O HNEGQIOMVPPMNR-IHWYPQMZSA-N 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007819 clotting time assay Methods 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 229940105772 coagulation factor vii Drugs 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 125000001995 cyclobutyl group Chemical group [H]C1([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-M deoxycholate Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-M 0.000 description 1
- 229940009976 deoxycholate Drugs 0.000 description 1
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 229940043237 diethanolamine Drugs 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 description 1
- 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 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 208000009190 disseminated intravascular coagulation Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-N dithiophosphoric acid Chemical class OP(O)(S)=S NAGJZTKCGNOGPW-UHFFFAOYSA-N 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000005712 elicitor Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000004049 epigenetic modification Effects 0.000 description 1
- AFAXGSQYZLGZPG-UHFFFAOYSA-N ethanedisulfonic acid Chemical compound OS(=O)(=O)CCS(O)(=O)=O AFAXGSQYZLGZPG-UHFFFAOYSA-N 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 1
- 239000005038 ethylene vinyl acetate Substances 0.000 description 1
- 229940012017 ethylenediamine Drugs 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000006624 extrinsic pathway Effects 0.000 description 1
- 229940012413 factor vii Drugs 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000003527 fibrinolytic agent Substances 0.000 description 1
- LRBQNJMCXXYXIU-QWKBTXIPSA-N gallotannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@H]2[C@@H]([C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-QWKBTXIPSA-N 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- RFDAIACWWDREDC-FRVQLJSFSA-N glycocholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 RFDAIACWWDREDC-FRVQLJSFSA-N 0.000 description 1
- 125000003827 glycol group Chemical group 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 125000000592 heterocycloalkyl group Chemical group 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000000138 intercalating agent Substances 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000001985 kidney epithelial cell Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 150000002632 lipids Chemical group 0.000 description 1
- 108010013555 lipoprotein-associated coagulation inhibitor Proteins 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 229960002510 mandelic acid Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 108091092021 miR-2150 stem-loop Proteins 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- YZMHQCWXYHARLS-UHFFFAOYSA-N naphthalene-1,2-disulfonic acid Chemical compound C1=CC=CC2=C(S(O)(=O)=O)C(S(=O)(=O)O)=CC=C21 YZMHQCWXYHARLS-UHFFFAOYSA-N 0.000 description 1
- PSZYNBSKGUBXEH-UHFFFAOYSA-N naphthalene-1-sulfonic acid Chemical compound C1=CC=C2C(S(=O)(=O)O)=CC=CC2=C1 PSZYNBSKGUBXEH-UHFFFAOYSA-N 0.000 description 1
- KVBGVZZKJNLNJU-UHFFFAOYSA-N naphthalene-2-sulfonic acid Chemical compound C1=CC=CC2=CC(S(=O)(=O)O)=CC=C21 KVBGVZZKJNLNJU-UHFFFAOYSA-N 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 125000001893 nitrooxy group Chemical group [O-][N+](=O)O* 0.000 description 1
- QTNLALDFXILRQO-UHFFFAOYSA-N nonadecane-1,2,3-triol Chemical compound CCCCCCCCCCCCCCCCC(O)C(O)CO QTNLALDFXILRQO-UHFFFAOYSA-N 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 125000000913 palmityl 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])([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
- WLJNZVDCPSBLRP-UHFFFAOYSA-N pamoic acid Chemical compound C1=CC=C2C(CC=3C4=CC=CC=C4C=C(C=3O)C(=O)O)=C(O)C(C(O)=O)=CC2=C1 WLJNZVDCPSBLRP-UHFFFAOYSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 150000008298 phosphoramidates Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 229920001200 poly(ethylene-vinyl acetate) Polymers 0.000 description 1
- 229920001515 polyalkylene glycol Polymers 0.000 description 1
- 239000010318 polygalacturonic acid Substances 0.000 description 1
- 229920002643 polyglutamic acid Polymers 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000008742 procoagulation Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000004088 pulmonary circulation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 210000005000 reproductive tract Anatomy 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 150000003873 salicylate salts Chemical class 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000001743 silencing effect Effects 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 108091029842 small nuclear ribonucleic acid Proteins 0.000 description 1
- RGHFKWPGWBFQLN-UHFFFAOYSA-M sodium;5,5-diethylpyrimidin-3-ide-2,4,6-trione Chemical compound [Na+].CCC1(CC)C([O-])=NC(=O)NC1=O RGHFKWPGWBFQLN-UHFFFAOYSA-M 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000001324 spliceosome Anatomy 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- IIACRCGMVDHOTQ-UHFFFAOYSA-N sulfamic acid Chemical group NS(O)(=O)=O IIACRCGMVDHOTQ-UHFFFAOYSA-N 0.000 description 1
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 1
- 150000003456 sulfonamides Chemical group 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 150000003457 sulfones Chemical group 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 108700004027 tat Genes Proteins 0.000 description 1
- 101150098170 tat gene Proteins 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- ZEMGGZBWXRYJHK-UHFFFAOYSA-N thiouracil Chemical compound O=C1C=CNC(=S)N1 ZEMGGZBWXRYJHK-UHFFFAOYSA-N 0.000 description 1
- 229960000103 thrombolytic agent Drugs 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000759 toxicological effect Toxicity 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- ZMANZCXQSJIPKH-UHFFFAOYSA-O triethylammonium ion Chemical compound CC[NH+](CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-O 0.000 description 1
- 125000000876 trifluoromethoxy group Chemical group FC(F)(F)O* 0.000 description 1
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 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
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 101150003485 unc-22 gene Proteins 0.000 description 1
- 125000002948 undecyl 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])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000011144 upstream manufacturing Methods 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
- 210000000626 ureter Anatomy 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
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
- C12N15/1138—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
- 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
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- 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
Definitions
- the present invention relates to synthesised RNAs, more specifically short interfering RNAs (siRNAs) that are able to modulate the expression of Tissue Factor (TF) and the use thereof in the prevention of metastasis and treatment of cancer.
- siRNAs short interfering RNAs
- the present invention also discloses novel siRNA molecules directed towards murine TF and the use thereof.
- RNA silencing is a new field of research that has coalesced during the last decade from independent studies on various organisms. It has been known for a long time that interactions between homologous DNA and/or RNA sequences can silence genes and induce DNA methylation (Bernstein E, Caudy AA, Hammond SM, Harmon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366 (2001). The discovery of RNA interference
- RNAi double-stranded RNA
- dsRNA double-stranded RNA
- RNAi posttranscriptional gene- silencing
- RNAi in animals, and the related phenomena of PTGS in plants, result from the same highly conserved mechanism, indicating an ancient origin (Bernstein E. et al. (2001), supra).
- the basic process involves a dsRNA that is processed into shorter units (called short interfering RNA; siRNA) that guide recognition and targeted cleavage of homologous messenger RNA (mRNA).
- siRNA short interfering RNA
- mRNA homologous messenger RNA
- the dsRNAs that (after processing) trigger RNAi/PTGS can be made in the nucleus or cytoplasm in a number of ways.
- the processing of dsRNA into siRNAs, which in turn degrade mRNA, is a two- step RNA degradation process.
- the first step involves a dsRNA endonuclease (ribonuclease Ill-like; RNase Ill-like) activity that processes dsRNA into sense and antisense RNAs which are 21 to 25 nucleotides (nt) long, i.e. siRNA. In Drosophila.this RNase Ill-type protein is termed Dicer.
- the antisense siRNAs produced combine with, and serve as guides for, a different ribonuclease complex called RNA-induced silencing complex (RISC), which cleaves the homologous single-stranded mRNAs.
- RISC RNA-induced silencing complex
- RISC cuts the mRNA approximately in the middle of the region paired with the antisense siRNA, after which the mRNA is further degraded.
- dsRNAs from different sources can enter the processing pathway leading to RNAi/PTGS.
- RNAi/PTGS RNAi/PTGS.
- recent work also suggests that there may be more than one pathway for dsRNA cleavage producing distinct classes of siRNAs that may not be functionally equivalent.
- RNA silencing (which is active at different levels of gene expression in the cytoplasm and the nucleus) appears to have evolved to counter the proliferation of foreign sequences such as transposable elements and viruses (many of which produce dsRNA during replication).
- foreign sequences such as transposable elements and viruses (many of which produce dsRNA during replication).
- RNAi/PTGS produce a mobile signal that induces silencing at distant sites, the possibility of injecting directly siRNAs to shut down protein synthesis and/or function as a therapeutic tool in mammalian cells should be considered.
- Garrus et al Garrus, J.E., von Schwedler, U.K., Pornillos, O.W., Morham, S.G., Zavitz, K.H., Wang, H.E., Wettstein, D.A., Stray, K.M., Cote, M., Rich, R.L., Myszka, D.G., Sundquist, W.I. (2001), "TsglOl and the vacuolar protein sorting pathway are essential for HIV-1 budding.”, Cell, 107:55-65), using 5, 6 and 7 mutations respectively.
- a central, double mutations used by Boutla and our own group (Boutla et al.
- shRNAs short hairpin RNAs induce sequence-specific silencing in mammalian cells.
- This shRNA construct was inactivated either by a single mutation in the putative second nucleotide of the shRNA, or by a single mismatch in the putative ninth nucleotide. Gitlin et al (Gitlin, L., Karelsky, S., Andino, R. (2002), "Short interfering RNA confers intracellular antiviral immunity in human cells",.
- siRNA resistant polio virus strains containing a single mutation in the target site on the genomic RNA either in the sixth nucleotide of the siRNA or the ninth nucleotide, both counted from the 5' end of the sense strand.
- siRNA seem to be inactivated to different degrees.
- chemical modification of nucleic acids has inter alia been used to protect single stranded nucleic acid sequences against nuclease degradation and thus obtaining sequences with longer half life.
- WO 91/15499 discloses 2'O-alkyl oligonucleotides useful as antisense probes.
- 2-O- methylation has been used to stabilize hammerhead ribozymes (Amarzguioui M,
- Tissue Factor and metastasis At present, cancer remains a major cause of death and this is often a consequence of metastasis.
- tumour colonies are established by malignant cells, which have detached from the original tumour (primary tumour) and spread throughout the body.
- the formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumour, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumour at the target site depends on angiogenesis.
- Tissue Factor is a membrane-bound glycoprotein mainly known as a potent trigger of blood coagulation (SNr E, Kolsto AB, Prydz H., "Cell biology of tissue factor, the principal initiator of blood coagulation.”, Thromb Res. 81, 1-41 (1996).) and instrumental in causing arterial thrombosis upon rupture of atherosclerotic plaques.
- TF is not found soluble in the circulation or accessible to plasma proteins including factor Vll/VIIa and the other coagulation factors. Expression of TF in the vascular compartment typically results in disseminated intravascular coagulation or localized initiation of clotting.
- tissue factor inhibitors have metastasis reducing abilities (Hu and Garen (2001), "Targeting tissue factor on tumor vascular endothelial cells and tumor cells for immunotherapy in mouse models of prostatic cancer.”, Proc.Natl.Acad.Sci, 98 (21): 10180-12185, A. Amirkhosravi et al. (2002), “Tissue Factor Pathway Inhibitor Reduces Experimental Lung Metastasis of B16 Melanomas.”, Thromb. Haemost., 87: 930-936).
- siRNA in silencing TF.
- Patent application WO 01/75164 discloses a Drosophila in vitro system which is used to demonstrate that dsRNA is processed to RNA segments 21-23 nucleotides (nt) in length, wherein these 21-23 nt fragments are specific mediators of RNA degradation.
- Caplen et al. reports that synthetic siRNA directed towards the CAT gene and C. elegans unc-22 gene reduced the expression in vertebrate and invertebrate systems respectively (Caplen, N.J. et al. (2001), "Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.”, Proc. Natl. Acad. Sci. USA, 98: 17, 9742-47).
- dsRNA is already established as a routine tool for gene silencing in e.g. plants, C elegans and Drosophila (Clemens, J. C. et al., "Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways.”, Proc. Natl Acad. Sci. USA 97, 6499-6503 (2000).
- dsRNA cannot be used in mammalian cells because of unspecified effects.
- sites on the mRNA target can also be differentially accessible to ribozymes (Amarzguioui M., Brede G. Babaie E., Grotli M., Sproat B., Prydz H., "Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes", Nucleic Acid Res., 28, 4113-4124 (2000)), efforts to identify really efficient ribozymes towards TF with little or no toxicity, have not yet succeeded.
- siRNA molecules directed towards TF modulate the activity of TF and that the TF reducing activity is highly sequence specific (PCT/NO03/00045, Holen, T. et al. (2000), "Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor", Nucleic Acid Res., 30, 1757-1766).
- PCT/NO03/00045 Holen, T. et al. (2000), "Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor", Nucleic Acid Res., 30, 1757-1766.
- the use of siRNA to inhibit TF and thus prevent metastasis would constitute a promising step forward in the cancer therapy.
- the present invention relates to short interfering RNA molecules which are double or single stranded and comprise at least 19 nucleotides, and wherein said siRNAs are able to modulate the gene expression of TF.
- siRNAs are dsRNAs of « 21-25 nucleotides that have been shown to function as key intermediates in triggering sequence-specific RNA degradation.
- siRNAs towards TF can bypass the RNAse Ill-like RNAi initiator Dicer and directly charge the effector nuclease RISC so that TF mRNA is degraded (PCT/NO03/00045). It was also demonstrated that different siRNAs against the same target vary in efficiency, and thus, siRNAs may be synthesised against different parts of TF mRNA, after which they combine with RISC which is then guided for specific degradation silencing of TF mRNA.
- siRNA molecules directed towards TF reduce malignant cells abilities to settle and form new tumours in vivo in a mouse model.
- the metastasis reducing effect of siRNA targeting TF is also demonstrated after systemic injection of siRNA.
- the siRNA molecules directed towards TF may be useful in the prevention of metastasis and treatment of cancer in vertebrates, preferably mammals, more preferably humans.
- the present inventions relates to the use of short interfering RNA molecules (siRNAs) directed towards TF for the preparation of a pharmaceutical composition for preventing metastasis.
- the present invention relates to the use of double or single stranded siRNA directed towards, a tissue factor (TF) coding nucleic acid sequence or fragments thereof, and wherein the siRNA molecule is selected from the group consisting of (a) a siRNA molecule having the nucleic acid sequence depicted in SEQ ID NO 1 to SEQ ID NO 8 or SEQ ID NO 32 to SEQ ID NO 37, the complement of which is SEQ ID NO 48 - SEQ ID 53; (b) a siRNA molecule having a sequence which is about 90 % homologous to a siRNA molecule of (a); (c) a siRNA molecule which compromise a sequence having a target site which is shifted up to 7 nucleotides in either the 5 ' or 3 ' terminal direction of the SEQ ID NO 1 to SEQ ID NO 8 or SEQ ID NO 32 to SEQ ID NO 37, the complement of which is SEQ ID NO 48 - SEQ ID 53; (d) a siRNA molecule having a sequence which is about 90
- siRNA to prevent metastasis may provide a better and more efficient treatment of cancer and preferably lead to increased survival rate.
- the said siRNAs are double stranded.
- siRNAs induces cleavage of TF mRNA, more preferably identified by SEQ ID NO 1 or SEQ ID NO 2.
- said composition comprise siRNAs which are 21-25 nucleotides long, more preferably 21 nucleotides long and even more preferably identified by SEQ ID NO 1 to SEQ ID NO 8.
- the siRNAs are directed to TF or fragments thereof, which are of vertebrate origin, preferably mammalian origin, more preferably human origin.
- the siRNA molecules comprises a sequence which is about 90 % homologous to a siRNA molecule depicted in SEQ ID NO 1 to SEQ ID NO 8 or that the siRNA comprises a sequence as depicted in SEQ ID NO 1 to SEQ ID NO 8 wherein a C]-C 3 -alkyl, C C 3 -alkenyl or Ci-C alkylyl group is introduced in one or more of the 2' OH hydroxyl group.
- the siRNA molecule has the sequence as depicted in SEQ ID NO 9 to SEQ ID NO 11 ).
- said siRNA molecules comprise a sequence as depicted SEQ ID NO 1 to SEQ ID NO 8, wherein the phosphodiester bond has been replaced by a thiophosphodiester bond.
- the modified sequence is the sequence SEQ ID NO 24, the complement of which is SEQ ID NO 40, SEQ ID NO 28, the complement of which is SEQ ID NO 44 or SEQ ID NO 29, the complement of which is SEQ ID NO 45.
- the present invention also provides novel murine siRNA sequences useful as e.g. a research tool for studying the mechanism of metastasis and the role of TF.
- the siRNA's have the nucleic acid sequences depicted in SEQ ID NO 32 to SEQ ID NO 37, the complement of which is SEQ ID NO 48 to SEQ ID NO 53, respectively.
- composition prepared according to the present use may furthermore comprise e.g. diluents, lubricants, binders, carriers, disintegration means, absorption means, colourings, sweeteners and/or flavourings. It is also favourable that the composition comprises adjuvants and/or other therapeutically principles. Further, in still another aspect, the said composition may be administered e.g. parenterally (e.g. by subcutaneous, intravenous, intramuscular or intraperitoneal injection or infusion), orally, nasally, buccally, rectally, vaginally and/or by inhalation or insufflation. More preferably, the composition may be formulated as e.g.
- compositions may be administered in one dose, in divided doses or by way of sustained release devices, preferably alone or together with other pharmaceuticals.
- the administration rout according to the method of the present invention is e.g. parenterally (e.g. by subcutaneous, intravenous, intramuscular or intraperitoneal injection or infusion), orally, nasally, buccally, rectally, vaginally and/or by inhalation or insufflations.
- double-stranded means a nucleic acid molecule having both a sense and an anti-sense strand.
- the sense strand and the antisense strand can be from the same nucleic acid molecule or assembled from two nucleic acid molecules and covalently connected via a linker molecule (e.g., a polynucleotide linker or a non-nucleotide linker).
- nucleic acid molecule means any chain of nucleotides or nucleic acid mimetics. Included in this definition are natural and non-natural oligonucleotides, both modified and unmodified.
- pharmaceutically acceptable carrier a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered.
- One exemplary pharmaceutically acceptable carrier substance is physiological saline.
- physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington 's Pharmaceutical Sciences, (20 th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA.
- reduce or inhibit means the ability to cause an overall decrease, preferably of 20% or greater, more preferably of 50% or greater, and most preferably of 75% or greater, in the level of protein or oligonucleotide as compared to a reference sample (e.g., a sample not treated with siRNA). This reduction or inhibition of RNA or protein expression can occur through targeted mRNA cleavage or degradation.
- Assays for protein expression or nucleic acid expression are known in the art and include, for example, ELISA, western blot analysis for protein expression, Southern blotting or PCR for DNA analysis, and northern blotting or RNase protection assays for RNA.
- reduce or inhibit is also meant an overall decrease preferably of 20% or greater, more preferably of
- RNA molecules small interfering RNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length that is used to identify a target gene or mRNA to be degraded.
- siRNAs can also include short hairpin RNAs (shRNA) in which both strands of an siRNA duplex are included within a single RNA molecule.
- shRNA short hairpin RNAs
- Double- stranded siRNAs generally consist of a sense and anti-sense strand.
- Single- stranded siRNAs generally consist of only the anti-sense strand that is complementary to the target gene.
- siRNA includes any form of RNA, preferably dsRNA (proteolytically cleaved products of larger dsRNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA) as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides.
- alterations can include the addition of non-nucleotide material, such as to the end(s) of the 21 to 23 nucleotide RNA or internally (at one or more nucleotides of the RNA).
- the RNA molecule contains a 3 'hydroxyl group.
- Nucleotides in the RNA molecules of the present invention can also comprise non- standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides.
- the double-stranded oligonucleotide may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.
- modified siRNAs Collectively, all such altered RNAs are referred to as modified siRNAs.
- siRNAs of the present invention need only be sufficiently similar to natural RNA such that it has the ability to mediate RNAi.
- RNAi refers to the ability to distinguish or identify which RNAs are to be degraded.
- RNAi is capable of decreasing the expression of TF in a cell by at least 10%, 20%, 30%, or 40%, more preferably by at least 50%, 60%, or 70%, and most preferably by at least 75%, 80%, 90%, 95% or more.
- short 21, 22, 23, 24, or 25 nucleotide double stranded RNAs are used to down regulate TF expression.
- Such RNAs are effective at down-regulating gene expression in mammalian tissue culture cell lines (Elbashir et al., Nature 411 :494-498, 2001, hereby incorporated by reference).
- RNA as used herein is meant an RNA comprising a duplex region complementary to an mRNA.
- a short hairpin RNA may comprise a duplex region containing nucleotides, where the duplex is between 19 and 29 bases in length, and the strands are separated by a single- stranded 3, 4, 5, 6, 7, 8, 9, or 10 base linker region.
- the linker region is 6 bases in length.
- tissue factor protein as used herein is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation) that is substantially identical to any mammalian TF or TF precursor molecule. See, for example, GenBank accession numbers AAH11029 (human), NP001984 (human), P20352 (mouse), AAH24886 (mouse), AAH16397 (mouse), P42533 (rat), P30931 (bovine), Q9JLU8 (guinea pig).
- TF is an integral membrane glycoprotein that can trigger blood coagulation via the extrinsic pathway (Bach et al., J. Biol Chem.
- TF consists of a protein component (previously referred to as TF apoprotein-III) and a phospholipid (Osterud and Rapaport, Proc. Natl. Acad. Sci. 74, 5260-5264 (1977)). TF from various organs and species has been reported to have a relative molecular mass of 42,000 to 53,000. Purification of TF has been reported from various tissues such as human brain (Guha et al. Proc. Natl. Acad. Sci-. 83, 299- 302 (1986) and Broze et al., J. Biol. Chem. 260, 10917-10920 (1985)); bovine brain (Bach et al, J.
- TF includes TF protein from any of the species or tissues described herein having TF biological activity.
- TF biological activity can be measured by any of several assays known in the art. Non-limiting examples include in vitro coagulation assays, one-stage clotting assays, two-stage clotting assays (Pitlick and Nemerson, Methods En ⁇ ymol, 45: 37-48 (1976)), TF clotting time assay (Santucci et al., Thromb. Haemost. 83:445-454, 2000), and prothrombin time assays.
- tissue factor nucleic acid is meant a nucleic acid molecule (e.g., DNA, cDNA, genomic, mRNA, RNA, dsRNA, antisense RNA, shRNA) substantially identical to any mammalian TF or TF precursor nucleic acid molecule or any nucleic acid molecule that encodes any of the TF proteins described above. See, for example, GenBank accession numbers Ml 6553 (human), BC011029 (human) NM01993 (human), AF540377 (human), U07619 (rat), M57896 (mouse), and M55390 (rabbit).
- treating means administering a compound or a pharmaceutical composition for prophylactic and/or therapeutic purposes.
- therapeutic treatment refers to administering treatment to a subject already suffering from a disease to improve the subject's condition.
- the subject is diagnosed as suffering from a coagulation disorder or a tumour with metastatic potential.
- prevent disease refers to prophylactic treatment of a subject who is not yet ill, but who is susceptible to, or otherwise at risk of, developing a particular disease.
- a subject is determined to be at risk of developing a coagulation disorder based on a family history of coagulation disorders or prior cardiac events.
- a subject is determined to be at risk of developing a tumour metastasis if the subject has been diagnosed with a malignant tumour.
- treating is the administration to a mammal either for therapeutic or prophylactic purposes.
- tumour an abnormal group of cells or tissue that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease.
- Tumours show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be either benign or malignant.
- Non-limiting examples of tumours include bladder, blood, bone, brain, breast, cartilage, colon, kidney,, liver, lung, lymph node, nervous tissue, ovarian, pancreatic, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testicular, thymus, thyroid, trachea, uro genital tract, ureter, urethrea, uterine, and vaginal tumours.
- metastasis is meant the spread of cancer cells from its original site to another part of the body.
- the formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumour, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumour at the target site depends on angiogenesis. Tumour metastasis often occurs even after the removal of the primary tumour because tumour cells or components may remain and develop metastatic potential.
- FIG. 1 siRNAs, reporter construct and RNAi of transgene expression; a) The sense (top) and antisense (bottom) strands of siRNA species targeting eight sites within human TF (Genbank entry Ace. No. M16553) mRNA are shown, b) Luciferase reporter construct of human TF and c) RNAi by siRNA in cotransfection assays (averages of three or more independent experiments each in triplicate, ⁇ s.d. are shown).
- Figure 2 Efficacy of the siRNAs in standard cotransfection assays in HaCaT cells. Different synthetic batches of the hTF167i siRNA showed similar efficacy. Results are averages of at least three experiments, each in triplicate.
- FIG. 3 siRNA mediated reduction of endogenous TF expression; a) hTF167i and hTF372i induced cleavage of mRNA in transfected cells.
- the Northern analysis of TF mRNA was performed after transfection of HaCaT cells with siRNA (100 nM) with GADPH as control. Arrowhead indicates cleavage fragments resulting from siRNA action, b) Measurements of the effect of siRNAs on steady state mRNA levels (filled bars), procoagulant activity (dotted bars) and TF protein (antigen) expression (hatched bars) show that siRNA reduces mRNA, TF antigen levels and procoagulant activity.
- procoagulant activity and antigen cells were harvested 48 h after si transfection to accommodate the 7-8 h half-life of TF protein. Data are from a representative experiment in triplicate.
- FIG. 5 Time-dependence of siRNA-mediated RNAi; a) Inhibitory activity is reduced when mutations (Ml and M2 refer to one and two mutations, respectively) are introduced into the siRNAs.
- Cells were transfected with 100 nM siRNA and harvested for mRNA isolation 4, 8, 24 and 48 h (filled bars, lined bars, white bars with black dots and hatched bars, respectively).
- Expression levels were no ⁇ nalised to GADPH and standardised to mock-transfected cells at all time-points, b) Time- course of decay of inhibitory effect for mRNA levels (closed diamonds), reporter gene activity (open triangles) and procoagulant activity (filled bars).
- Figure 6 siRNA modifications.
- A Mutated and wild type versions of the siRNA hTF167i.
- the sequence of the sense strand of wild type (wt) siRNA corresponds to position 167-187 in human Tissue Factor (Ass. No. M16553).
- Single (si, s2, s3, s4, s7, slO, sl l, sl3, sl6) and double mutants (ds 7/10, ds 10/11, dslO/13, dslO/16) are all named according to the position of the mutation, counted from the 5 'end of the sense strand. All mutations (in bold) are GC inversions relative to the wild type.
- Phosphorothioate linkages are indicated by asteriscs (*), while 2'- ⁇ 9-mefhylated and 2'-( -allylated ribonucleotides are in normal and underlined bold upper case, respectively.
- Figure 8 Activity of chemically modified siRNA against endogenous TF mRNA. Experiments were performed and analysed as described in figure 7.
- Figure 9 Persistence of TF silencing by chemically modified siRNAs.
- Figure 10 shows the effect of i.v. injection of TF siRNA-transfected B 16 cells in lungs of C57 BL/6 mice.
- Figure 11 shows the effect of systemic application of siRNA.
- Mice in the control group received one i.v. injection with B16 melanoma cells.
- Mice in the test group received additionally three i.p. injections of siRNA targeting TF. These injections were done 1 day before, and 3 and 6 days after injection of the cells.
- RNAi is a form of post-transcriptional gene silencing initiated by the introduction of siRNAs. Short 21 to 25 nucleotide double-stranded RNAs are effective at down-regulating gene expression in nematodes (Zamore et al., Cell 101 :25-33, 2000) and in mammalian tissue culture cell lines (Elbashir et al., Nature 411 :494- 498, 2001). The further therapeutic effectiveness of this approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418:38-39, 2002).
- the nucleic acid sequence of a mammalian gene, such as TF can be used to design small interfering RNAs (siRNAs) that will inactivate TF target genes that have the specific 21 to 25 nucleotide RNA sequences used.
- siRNAs that target TF may be used, for example, as therapeutics to treat or prevent a coagulation disorder or a metastatic tumour.
- siRNAs may be designed to inactivate target genes of interest and screened for effective gene silencing, as described herein.
- additional siRNAs may be designed using standard methods.
- Short hairpin RNAs shRNAs
- shRNAs can also be used for RNAi as described in Paddison et al. (Proc. Natl. Acad. Sci USA, 99:6047-6052, 2002; Genes & Dev, 16:948-958, 2002).
- siRNA and shRNA candidate sequences are identified by empirical testing.
- One strategy for such testing is to construct a large library of non- overlapping synthetic siRNAs or shRNA encoding vectors that give good coverage of a tissue factor gene of interest, according to its largest sequenced cDNA, which includes partial 5' and 3 'UTR sequences.
- target knock-down such as Taqman quantitative RT-PCR and ELISA assays
- the process of siRNA or shRNA selection is relatively straightforward once conditions have been optimized for transfection and target measurements.
- a nucleoside is a nucleobase-sugar combination.
- the base portion of the nucleoside is normally a heterocyclic base.
- the two most common classes of such heterocyclic bases are the purines and the pyrimidines..
- Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
- the phosphate group can be linked to either the 2', 3 ' or 5' hydroxyl moiety of the sugar.
- the phosphate groups covalently link adjacent nucleosides to one another to fo ⁇ n a linear polymeric compound.
- the respective ends of this linear polymeric structure can be further joined to fo ⁇ n a circular structure; open linear structures are generally preferred.
- the phosphate groups are commonly referred to as fonning the backbone of the oligonucleotide.
- the nonnal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
- siRNA molecules used according to the present invention preferably include oligonucleotides containing modified backbones or non-natural internucleoside linkages.
- oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
- modified nucleobase oligomers that do not have a phosphorus atom in their internucleoside backbone are also considered to be nucleobase oligomers.
- Non-limited examples of nucleobase oligomers having modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphor amidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having no ⁇ nal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity, wherein the adjacent pairs of nucleoside units are linked 3 '-5' to 5 '-3' or 2 '-5' to 5 '-2'.
- Nucleobase oligomers having modified backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
- morpholino linkages formed in part from the sugar portion of a nucleoside
- siloxane backbones sulfide, sulfoxide and sulfone backbones
- fo ⁇ nacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
- alkene containing backbones sulfamate backbones
- sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
- PNA Peptide Nucleic Acids
- PNA compounds contain an amide backbone, more specifically an aminoethylglycine backbone.
- the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Methods for making and using these nucleobase oligomers are described, for example, in "Peptide Nucleic Acids: Protocols and Applications” Ed. P.E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999. Methods for the preparation of PNAs are disclosed e.g in U.S. Patent Nos.: 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference.
- the nucleobase oligomers have phosphorothioate backbones and nucleosides with heteroatom backbones, and in particular -CH 2 -NH-O-CH 2 -, -CH 2 -N(CH 3 )-O-CH 2 - (known as a methylene (methylimino) or MMI backbone), -CH 2 -O-N(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )- CH 2 -, and -O-N(CH 3 )-CH 2 -CH 2 -.
- the oligonucleotides have morpholino backbone structures as described in U.S. Patent No. 5,034,506. Nucleobase oligomers may also contain one or more substituted sugar moieties.
- Nucleobase oligomers comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted Ci to Cj . o alkyl or C 2 to C 10 alkenyl and alkynyl.
- Other preferred nucleobase oligomers include one of the following at the 2' position: Cj .
- Preferred modifications are 2'-O- methyl and 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2- methoxyethyl) or 2'-MOE).
- Another desirable modification is 2'- dimethylaminooxyethoxy (i.e., O(CH 2 ) 2 ON(CH 3 ) 2 ), also known as 2'-DMAOE.
- Other modifications include, 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2'- fluoro (2'-F).
- Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
- Oligonucleotides may also include nucleobase modifications or substitutions.
- "unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
- Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-halouracil and cytosine; 5- propynyl uracil and cytosine; 6-azo uracil, cytosine and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo (e.g., 5-bromo), 5- trifluorom e
- nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of an antisense oligonucleotide of the invention.
- 5-substituted pyrimidines include 5-substituted pyrimidines, 6- azapyrimidines, and N-2, N-6 and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
- 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 degrees Celsius per base pair. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are desirable base substitutions, even more particularly when combined with 2'-O-methoxyethyl or 2'-O-methyl sugar modifications.
- nucleobase oligomer of the invention involves chemically linking to the nucleobase oligomer one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
- moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553- 6556, 1989), cholic acid (Manoharan et al., Bioorg. Med. Chem.
- a thioether e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-
- the present invention also includes nucleobase oligomers that are chimeric compounds.
- "Chimeric" nucleobase oligomers are nucleobase oligomers, particularly oligonucleotides, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide.
- These nucleobase oligomers typically contain at least one region where the nucleobase oligomer is modified to confer, upon the nucleobase oligomer, increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
- An additional region of the nucleobase oligomer may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
- RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of nucleobase oligomer inhibition of gene expression. Consequently, comparable results can often be obtained with shorter nucleobase' oligomers when chimeric nucleobase oligomers are used, compared to phosphorothioate oligodeoxynucleotides hybridizing to the same target region.
- Chimeric nucleobase oligomers of the invention may be formed as composite structures of two or more nucleobase oligomers as described above. Such nucleobase oligomers, when oligonucleotides, have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include U.S. Patent Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.
- nucleobase oligomers used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
- Equipment for such synthesis is sold by several vendors including, for example, Applied Bio systems (Foster City, Calif). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
- nucleobase oligomers of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
- suitable formulations provided for assisting uptake, distribution and/or absorption assisting formulations: U.S.
- nucleobase oligomers of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound that, upon administration to an animal, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
- prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
- pharmaceutically acceptable salts refers to salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
- Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium 5 and the like. Examples of suitable amines are N,N'-dibenzylethylene- diamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylene- diamine, N-methylglucamine, and procaine (see, for example, Berge et al., J. Pharma Sci., 66: 1-19, 1977).
- the base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
- the free acid form may be regenerated by contacting the salt fo ⁇ n with an acid and isolating the free acid in. the conventional manner.
- the free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.
- a "pha ⁇ naceutical addition salt” includes a pharmaceutically acceptable salt of an acid fo ⁇ n of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines.
- Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates.
- Other suitable pharmaceutically acceptable salts are well known to the person skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicy
- Suitable phannaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations.
- suitable phannaceutically acceptable salts include (i) salts fonned with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (ii) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (iii) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p- toluenesulfonic acid, naphthalened
- the present invention also includes phannaceutical compositions and fonnulations that include the nucleobase oligomers of the invention.
- the pha ⁇ naceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
- mutations of the siRNA molecules directed towards TF are also included in the present invention.
- Preferred mutations include single base-pair mutations, including but not limited to those described in Example 5, and double base-pair mutations, also including but not limited to those described in Example 5.
- One way to simplify the manipulation and handling of the siRNA molecules is to place a cDNA cassette encoding the siRNA molecule under the control of a suitable promoter.
- the promoter must be capable of driving expression of the siRNA in the desired target host cell.
- the selection of appropriate promoters can readily be accomplished. Preferably, one would use a high expression promoter.
- suitable promoters include the self-contained polymerase III promoters U6 or HI, which are able to generate a transcript of defined sequence.
- An example of a suitable polymerase II promoter is the 763-base- ⁇ air cytomegalovirus (CMV) promoter.
- the recombinant vector can be a plasmid vector such as pUCl 18, pBR322, or other known plasmid vectors, that includes, for example, an E. coli origin of replication (see, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory press, 1989).
- the plasmid vector may also include a selectable marker such as the ⁇ lactamase gene for ampicillin resistance, provided that the marker polypeptide does not adversely affect the metabolism of the organism being treated.
- the cassette can also be bound to a nucleic acid binding moiety in a synthetic delivery system, such as the system disclosed in PCT Publication No. WO95/22618.
- the nucleic acid can be introduced into the cells by any means appropriate for the vector employed. Many such methods are well known in the art (Sambrook et al., supra, and Watson et al., "Recombinant DNA", Chapter 12, 2d edition, Scientific American Books, 1992). Recombinant vectors can be transfened by methods such as calcium phosphate precipitation, electroporation, liposome-mediated transfection, gene gun, microinjection, viral capsid-mediated transfer, polybrene- mediated transfer, or protoplast fusion.
- Transfer of the recombinant vector can be accomplished through direct injection into the amniotic fluid or intravenous delivery.
- Gene delivery using adenoviral vectors or adeno-associated vectors (AAV) can also be used.
- Adenoviruses are present in a large number of animal species, are not very pathogenic, and can replicate equally well in dividing and quiescent cells. As a general rule, adenoviruses used for gene delivery are lacking one or more genes required for viral replication.
- Replication-defective recombinant adenoviral vectors can be produced in accordance with art-known techniques (see Quantin et al., Proc. Natl. Acad. Sci.
- plasmid or viral vectors may contain, for example, a promoter, including, but not limited to the polymerase I, II, and III HI, U6, BL, SMK, 7SK, tRNA polIII, fRNA(met)-derived, and T7 promoters, a cloning site for the stem-looped RNA coding insert, and a 4-5- thymidine transcription tennination signal.
- a promoter including, but not limited to the polymerase I, II, and III HI, U6, BL, SMK, 7SK, tRNA polIII, fRNA(met)-derived, and T7 promoters, a cloning site for the stem-looped RNA coding insert, and a 4-5- thymidine transcription tennination signal.
- the Polymerase III promoters generally have well-defined initiation and stop sites and their transcripts lack poly(A) tails.
- the termination signal . for these promoters is defined by the poly- thymidine tract, and the transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3' UU overhang in the expressed shRNA, which is similar to the 3' overhangs of synthetic siRNAs.
- transfection reagents including but not limited to: TransIT-TKO3 (Minis, Cat. # MIR 2150), Trans messenger3 (Qiagen, Cat. # 301525), and Oligofectamine3 (Invitrogen, Cat. # MIR 12252-011). Protocols for each transfection reagent are - available from the manufacturer. Additional formulations that aid in the delivery of oligonucleotides or other nucleobase oligomers to cells are described in (see, e.g., U.S.
- Patents 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055) The concentration of siRNA used for each target and each cell line varies but in general ranges from 0.05 nM to 500 nM, more preferably 0.1 nM to 100 nM, and most preferably 1 nM to 50 nM. If desired, cells can be transfected multiple times, using multiple siRNAs to optimize the gene-silencing effect.
- siRNA siRNA
- RNA polymerase III transcription units which noraially encode the small nuclear RNA U6 or the human RNAse P RNA HI.
- These expression cassettes allow for the expression of both sense and anti-sense RNA.
- the endogenous expression of siRNA from introduced DNA templates is thought to overcome some limitations of exogenous siRNA delivery, in particular the transient loss of phenotype. In fact, stable cell lines have been obtained using these siRNA expression cassettes allowing for a stable loss of function phenotype (Miyagishi M.
- RNAi of TF can be generated using the above techniques.
- Assays for evaluating gene silencing effect mRNA and protein expression can be analyzed using any of a variety of art known methods including but not limited to northern blot analysis, RNAse protection assays, luciferase or ⁇ -gal reporter assays, western blots, and immunological methods such as ELISAs.
- the siRNAs according to the present invention can be used to down-regulate the expression or biological activity of mammalian TF.
- the siRNAs of the present invention can be used to treat or prevent tumour metastasis in a warm- blooded animal including, but not limited to, a human, cow, horse, pig, sheep, bird, mouse, rat, dog, cat, monkey, baboon, or the like.
- Treatment generally begins at a hospital so that the doctor can observe the therapy's effects closely and make any adjustments that are needed.
- the duration of the therapy depends on the tumour- metastasis being treated, the age and condition of the patient, the stage and type of the patient's disease, and how the patient's body responds to the treatment.
- Treatment may be performed at different intervals (e.g., daily, weekly, or monthly). Therapy may be given in on-and-off cycles that include rest periods so that the patient's body has a chance to build healthy new cells and regain its strength.
- Therapeutic treatments for metastatic tumours can be used to prevent tumour metastasis, slow the metastasis, slow the tumour-driven angiogenesis, to slow the tumour's growth, to kill or arrest tumour cells that may have spread to other parts of the body from the original tumour, or to relieve symptoms caused by the cancer.
- siRNA molecule of the invention may be administered together with a phannaceutically acceptable diluent, carrier, or excipient, in unit dosage fonn.
- Conventional phannaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from a disease that is caused by excessive cell proliferation. Administration may begin before the patient is symptomatic. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal, intracistemal, intraperitoneal, intranasal, aerosol, suppository, or oral administration.
- therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal fonnulations, in the fonn of powders, nasal drops, or aerosols.
- intravenous administration can be used to inject siRNAs directly into the blood stream to treat a coagulation disorder.
- direct injection of siRNA into tumours can be used to treat metastatic tumours.
- Fonnulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
- polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
- Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
- Other potentially useful parenteral delivery systems for tissue factor modulatory compounds include ethyl ene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
- Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
- the fonnulations can be administered to human patients in therapeutically effective amounts (e.g., amounts which prevent, eliminate, or reduce a pathological condition) to provide therapy for a disease or condition.
- therapeutically effective amounts e.g., amounts which prevent, eliminate, or reduce a pathological condition
- the preferred dosage of an siRNA molecule of the invention is likely to depend on such variables as the type and extent of the disorder, the overall health status of the particular patient, the foraiulation of the compound excipients, and its route of administration.
- the dosage of administered siRNAs will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history, disease progression, tumour burden, and the like.
- the dose is administered as indicated.
- Other therapeutic drugs may be administered in conjunction with the siRNA molecules.
- the pha ⁇ naceutical composition used for the treatment of tumours may optionally contain other chemotherapeutic agents, antibodies, antivirals, exogenous immunomodulators or the like.
- the pharmaceutical composition used for the treatment of coagulation disorders may optionally contain additional thrombolytic agents or anticoagulants such as heparin.
- the efficacy of treatment using the siRNAs described herein may be assessed by determination of alterations in the expression, concentration, or biological activity of the DNA, RNA or gene product of TF; clot dissolution; clot prevention; tumor regression; metastasis regression; metastasis prevention; or a reduction of the pathology or symptoms associated with the tumour.
- siRNAs In order to provide the siRNAs to obtain silencing of human TF (hTF), 21- nucleotide RNAs were chemically synthesised using phoshoramidites (Pharmacia and ABI) as described in PCT/NO03/00045. Thirteen siRNAs against hTF mRNA (Wiiger MT, Pringle S, Pettersen KS, Narahara N, Prydz H. Effects of binding of ligand (FVIIa) to induced tissue factor in human endothelial cells. Thromb Res. 98, 311-321 (2000) were synthesised (Fig. la). Eight of these siRNAs are termed SEQ ID NO 1 to SEQ ID NO 8, respectively.
- the present invention relates inter alia to the use of the synthesised siRNAs according to SEQ ID NO 1 to SEQ ID NO 31 disclosed in PCT/NO03/00045.
- the invention relates to novel murin siRNA molecules and the use thereof, specifically siRNA sequences having the nucleic acid sequence depicted in SEQ ID NO 32 to SEQ ID NO 37, the complement of which is SEQ ID NO 48 to SEQ ID NO 53, respectively.
- siRNAs directed toward TF have also been mapped more systematically. To avoid affecting the duplex stability of the siRNA, only GC pairs were targeted for mutation, by inversion of the pairs as described in example 5 below.
- the reporter constructs of human TF to be used in the Dual Luciferase system were designed using the coding region of TF which were cloned in- frame with the Firefly luciferase (LUC) gene, producing the fusion construct TF- LUC (Ace. No. AF416989). Numbering of the fusion construct refers to that of the genbank entry for TF and to the pGL3-enhancer plasmid (Promega) for LUC.
- the plasmid pcDNA3-Rluc (Ace. No. AF416990), encoding Renilla luciferase (Rluc; not shown) was used as internal control. Regions of TF and LUC cDNA contained within the construct are indicated in Figure lb.
- the Dual Luciferase system is a reporter system which is used to detect how much TF mRNA that is degraded by siRNA(s).
- HeLa, Cos-1 and 293 cells were maintained in Dulbecco's Minimal Essential Medium (DMEM) supplemented with 10%> fetal calf serum (Gibco BRL).
- DMEM Dulbecco's Minimal Essential Medium
- the human keratinocyte cell line HaCaT was cultured in serum free keratinocyte medium supplemented with 2,5 ng/ml epide ⁇ nal growth factor and 25 ⁇ g/ml bovine pituitary extract. All cell lines were regularly passaged at sub-confluence. The day before the experiment cells cultured in DMEM were trypsinized and resuspended in full medium before plating. HaCaT cells were trypsinized until detachment.
- HaCaT cells in 6-well plates were transfected with 100 nM siRNA in serum-free medium. Lipofectamine2000TM was used for higher transfection efficiency.
- Poly(A) mRNA was isolated 24 h after transfection using Dynabeads oligo(dT) 25 (Dynal). Isolated mRNA was fractionated for 16-18 h on 1,3% agarose/formaldehyde (0,8 M) gels and blotted on to nylon membranes (MagnaCharge, Micron Separations Inc.). Membranes were hybridised with random-primed TF (position 61-1217 in cDNA) and GAPDH (1,2 kb) cDNA probes in PerfectHyb hybridisation buffer (Sigma) as recommended by the manufacturer.
- TF corresponds to 1,5 ng TF as detennined in the TF ELISA (Wiiger MT et al., (2000), supra, and discardr E. et al., (1996), supra).
- the activity was normalised to the protein content in the cell homogenates, as measured by the BioRad DC assay.
- TF antigen was quantified using the Imubind Tissue Factor ELISA kit (American Diagnostica, Greenwich, CT, USA). This ELISA recognises TF apoprotein, TF and TF: Coagulation Factor VII (FVII) complexes. The samples were left to thaw at 37°C and homogenised. An aliquot of each homogenate (100 ⁇ l) was diluted in phosphate-buffered saline containing 1% BSA and 0,1% Triton X-100. This sample was then added to the ELIS A-well and the procedure from the manufacturer followed. The antigen levels were no ⁇ nalised to the total protein content in the cell homogenates. All the various mutant siRNAs were analysed for depletion of endogenous TF mRNA in HaCaT cells, 24h after LIPOFECTAMINE2000-mediated transfection, as described previously for the wild type siRNA sequences.
- FVII Coagulation Factor VII
- the preparation of a phannaceutical composition according to the present use of the invention may be provided by using techniques well-known to the person skilled in the art.
- the composition may comprise one or more of said siRNAs, and optionally diluents, lubricants, binders, earners disintegration and/or absorption means, colourings, sweeteners flavourings etc., all known in the art.
- Furthennore the said composition may also comprise adjuvants and/or other therapeutical principles, and may be administered alone or together with other pharmaceuticals.
- Said composition may be used before, simultaneous or after conventional cancer treatment regimes, e.g. cytostatica treatments, radiation etc.
- Said composition may also be used before, simultaneous or after surgical intervention, e.g.
- a pharmaceutical composition prepared according to the present use may be administered e.g. parenterally (e.g. by subcutaneous, intravenous, intramuscular or intraperitoneal injection or infusion of sterile solutions or suspensions), orally (e.g. in the form of capsules, tablets, pills, suspensions or solutions), nasally (e.g. in fonn of solutions/spray), buccally, rectally (e.g. in the form of suppositories), vaginally (e.g. in the fonn of suppositories), by inhalation or insufflation (e.g.
- parenterally e.g. by subcutaneous, intravenous, intramuscular or intraperitoneal injection or infusion of sterile solutions or suspensions
- orally e.g. in the form of capsules, tablets, pills, suspensions or solutions
- nasally e.g. in fonn of solutions/spray
- buccally e.g. in the form of sup
- pha ⁇ naceutical composition in the form of an aerosol or solution/spray), via an implanted reservoir, or by any other suitable route of administration, in dosage fonnulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and/or vehicles.
- the pha ⁇ naceutical composition may further be administered in one dose, in divided doses or by way of sustained release devices.
- siRNAs according to SEQ ID NO 1 to SEQ ID NO 31 and the novel murine TF (mTF) siRNA sequences according to SEQ ID NO 32 to SEQ ID NO 37 were chemically synthesised according to the method described in PCT/NO03/00045. Double-stranded siRNA complementary to a certain partial sequence on the targeted TF mRNA sequence induces degradation of this specific mRNA in mammalian cells (see Example 1). This effect was highly sequence-dependent, and contrary to data in lower organisms, as only a few sites on the TF mRNA were highly susceptible to the conesponding siRNAs.
- Example 2 the depletion of TF mRNA results in marked reduction of TF protein and procoagulant activity.
- All the mTF siRNA sequences target site lie within a 200 bp region corresponding to the region harbouring the best siRNA targets in hTF (cf. PCT/NO03/00045).
- three different target sites were designed against the locus corresponding to the hTF167i. Where possible, the positioning of the siRNA within each locus has been chosen to achieve the highest possible match with the human sequence.
- All siRNAs are synthesized with 2 bp target specific ribonucleotide overhangs (i.e. no DNA in the ends).
- SEQ ID NO 32-37 showed significantly metastasis reducing activity in a mouse model as described in Example 7.
- Example 1 Analysis of hTF siRNA efficacy in cells transiently cotransfected with hTF-LUC and hTF siRNA (i.e. analysis of RNAi by siRNA(s) in cotransfection assays)
- TF siRNA efficacy was perfonned in HeLa cells transiently cotransfected with hTF-LUC (Fig. lb) and hTF siRNA (Fig. la) using the Dual Luciferase system (Promega). Ratios of LUC to Rluc expression were normalised to levels in cells transfected with a representative irrelevant siRNA, Protein Serine Kinase 314i (PSK314i).
- PSK314i Protein Serine Kinase 314i
- siRNAs had potent and specific effects in the cotransfection assays, with the best candidates, hTF167i and hTF372i, resulting in only 10-15 % residual luciferase activity in HeLa cells (Fig. lc). Furthermore, also a positional effect was found, as hTF562i showed only intennediate effect, and hTF478i had very low activity. This pattern was also found in 293, COS-1 and HaCaT cells (Fig.lc), and with siRNAs from different synthetic batches and at various concentrations (the siRNAs caused the same degree of inhibition over a concentration range of 1-100 nM in cotransfection assays; data not shown).
- siRNAs (hTF158i, hTF161i, hTF164i, hTF170i, hTF173i and hTF176i) were synthesized which targeted sites shifted at both sides of hTF167i in increments of 3 nts, wherein each of them shared 18 out of 21 nts with its neighbours (see Fig. lc). Surprisingly it was found that despite the minimal sequence and position-differences between these siRNAs, they displayed a wide range of activities (Figure 2).
- the . present invention also relates to siRNA which is able to cleave mRNA in mammalian cells. Furthe ⁇ nore, the observed effect suggests that RISC may be active also in mammals. The third best.
- siRNA in cotransfection assays also resulted in significant depletion of TF mRNA levels (57% residual expression, data not shown).
- the remaining TF siRNA did not show any activity as measured by Northern assays (Fig. 3b), nor did they stimulate TF expression, a point of some interest, as transfection with chemically modified ribozymes can induce TF mRNA three-fold (data not shown).
- this relative inertness of irrelevant siRNAs i.e. siRNAs with «non-specific» effects
- the coagulation activity in the HaCaT cells was reduced 5-fold and 2-fold, respectively, in cells transfected with hTF167i and hTF372i, compared to mock- transfected cells (Fig.3b and Fig.5b).
- the effect of siRNAs on total cellular TF protein was also measured (Fig. 3b), and demonstrated an inhibitory effect that was generally greater than the observed effect on pro coagulation activity.
- the siRNAs hTF167i and hTF372i display specificity and potency in a complex physiological system, and that we have demonstrated positional effects, as other siRNA molecules against the same target mRNA are basically inactive.
- siRNA-induced target degradation was incomplete (a level of approximately 10% of the target mRNA remained even with the most effective siRNAs), may be due to the presence of a fraction of mRNA in a protected compartment, e.g. in spliceosomes or in other nuclear locations.
- a more likely possibility may be a kinetically determined balance between transcription and degradation, the latter being a time-consuming process.
- RNAi in C. elegans. Science 287, 2494-2497 (2000) have suggested the existence of a system whereby certain siRNA genes are involved in the heritability of induced phenotypes.
- the persistence of the siRNA silencing in HaCaT cells transfected at a very low cell density was measured.
- Fig. 5b The level of TF mRNA in mock- transfected control cells declined gradually during the experiment, in what appeared to be cell expansion-dependent down-regulation of expression.
- Example 5 Analysis of the effect of introducing base-pairing mutations in the siRNA sequences.
- siRNA were mapped more systematically in order to detennine whether mutations were equally tolerated within the whole siRNA.
- a total of 8 different new single-mutant siRNA were designed and named according to the position (starting from the 5' of the sense strand) of the mutation (si, s2, s3, s4, s7, sl l, sl3, sl6, i.e. SEQ ID NO 9- 17).
- the previously described central single-mutant Ml (example 4) was included in this analysis and renamed slO. All siRNAs were analysed for productive annealing by denaturing PAGE (15%).
- siRNAs with one modification each in the extreme 5' and 3' ends of the siRNA strands (Pl+1, Ml+1, Al+1, i.e. SEQ ID NO 22, the compliment of which is SEQ ID NO 38, SEQ ID NO 26, the compliment of which is SEQ ID NO 42 and SEQ ID NO 30, the compliment of which is SEQ ID NO 46, respectively) was initially synthesized.
- the 5' end of the chemically synthesized siRNAs might be more sensitive to modification since it has to be phosphorylated in vivo to be active (Nykanen, A., Haley, B. and Zamore, P.D. (2001) ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway. Cell, 107:309- 321).
- siRNAs PO+2, M0+2 and AO+2, i.e. SEQ ID NO 23, 27 and 31, respectively, cf. figure 6 which were known to be tolerant for various types of modifications (Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21 -nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498 (2001), Elbashir, S.M., Martinez,- J.,
- siRNAs were likewise analysed for initial activity 24h following transfection into HaCaT cells. Normalized expression levels in cells transfected with modified siRNAs ' were slightly elevated, at 16-18% residual levels compared to 1 1% in cells transfected with wild type.
- siRNA The most extensively phosphorothioated siRNA proved to be toxic to cells, resulting in approximately 70% cell death compared to mock- transfected cells (measured as the expression level of the control mRNA GAPDH). Due to these complications, this siRNA species was not included in further analysis. The remaining siRNA species were evaluated for increased persistence of silencing by analysing TF mRNA expression 5 days after a single transfection of lOOnM siRNA. At this point, TF expression in cells transfected with wild type siRNA had recovered almost completely (80% residual expression compared to mock-trans feet ed cells) (figure 9a).
- Example 7 siRNA toward murin TF reduces circulating malignant cells ability to form pulmonary tumours.
- mice Tissue Factor
- the experiments were designed and carried out to investigate if knockdown of Tissue Factor (TF) reduced the ability of tail vein - injected cells to settle in the pulmonary circulation and fo ⁇ n lung tumors in mice.
- The' cells were transfected with siRNA against murine TF before injection of the knockdown cells, which had a reduction in their TF level down to 10 - 20 percent of control mouse before injection.
- the mice were sacrificed 6 - 25 days after injection of the cells. In each mouse had 0.2 to 1.0 million cells in 0.2 ml medium been injected. All mice used were C57B1.
- Three groups were tested in each experiment: Group 1 : pretreatment of cells before injection was with siRNA against PSKH1, a serine kinase of unknown function.
- Group 2 pretreatment of the cells with siRNA against murine TF.
- Number of macroscopically visible tumours in the lungs was counted after autopsy of the mice. Examples of the results are given in Table 1 and 2.
- Mice receiving cells retreated with siRNA against murine TF (Group 2) develop a low number of tumours.
- Group 1 mice develop around 10 times more tumours than group 2, and mice of group 3 have more than 500 tumours in their lungs. Since the effect of siRNA on its target mRNA is highly specific (if the siRNA sequence has been properly selected to not bind other nucleotide sequences), the group 3 mice may have up to 200 - 250 - fold increase in their lung tumours when compared to mice in group 2.
- siRNA directed towards TF are useful in the preventions of metastasis and may be used in the treatment of cancer in mammals.
- Highly specific TF siRNA molecules adapted to the TF sequence of a specific species may be found by the screening method disclosed in PCT/NO04/00007.
- Table 1 Number of pulmonary metastases 10 days after injection of TF siRNA.transfected B16 cells in C57 mice.
- Table 2 Number of pulmonary metastases 15 days after injection of TF siRNA.transfected B16 cells in C57 mice.
- Example 8 siRNA toward murin TF reduces circulating malignant cells ability to form pulmonary tumours.
- siRNA specific for murine TF mTF
- Lipofectamine2000-mediated transfection of B16 cells with 100 nM siRNA demonstrated a highly variable efficiency of the different target sequences, consistent with previous observations (Holen, T. et al. (2002), supra).
- the two most effective siRNA, mTF223i and mTF321i consistently depleted mTF mRNA by 70-80% in cultured B 16 cells.
- Tail vein injections have been assumed to represent a model for tumor take of blood-borne metastases.
- B16 B16-F10
- B16 murine melanoma cells into C57BL/6 mice results in pulmonary colonization within 10-14 days.
- TF Tissue Factor
- the day before injection cells were transfected with a control siRNA against hTF (hTF167i) or with either of two different siRNA targeting mTF (mTF223i, mTF321i).
- hTF167i control siRNA against hTF
- mTF223i two different siRNA targeting mTF
- mice were harvested on day 10 in the first experiment, on days 10 and 15 in the second experiment, and on days 15 and 20 in the third experiment.
- the data from all experiments are summarized in Table 1.
- a picture of representative lungs from mice treated from the test and control groups harvested at days 10 and 15 is shown in Figure 10. Both groups of mice that were treated with cells transiently transfected with active (mTF) siRNA, and therefore exhibiting reduced expression of TF, developed significantly less tumors than the control group of mice at all time-points investigated.
- mTF active siRNA
- TF has a crucial function in promoting lung tumor metastasis of B16 melanoma cells in the C57BL/6 mice.
- siRNA directed towards TF is useful in the prevention of metastasis and may be used in the treatment of cancer in mammals.
- Highly specific TF siRNA molecules adapted to the TF sequence of a specific species may be found by the screening method disclosed in PCT/NO04/00007.
- example 7 and 8 it is demonstrated that knockdown of TF prevents colonization of the lungs by B16 melanoma cells.
- the inventors have furthennore demonstrated that that the effect of siRNA targeting TF is evident also after systemic injection of siRNA, thus allowing potential therapeutic use of the siRNA (cf figure 11).
- hTF167i was used as a negative control.
- An injection mixture was prepared for intraperitonal (i.p.) injection in a total volume of 15ml for each mixture of mTF siRNA and hTF siRNA: • 1050 ⁇ l annealed siRNA (50 ⁇ M, 50 ⁇ g/ml) + 6500 ⁇ l OptiMEM • 1500 ⁇ l Lipofectamine200 + 6000 ⁇ l OptiMEM
- mice were divided into groups of ten animals each.
- the groups received one i.v. injection with B16 melanoma cells.
- the test group additionally received i.p injections of the injection mixture comprising the TF targeting siRNA molecules to be tested.
- One injection were administered one day previous to the administration of the B16 melanoma cells, and additionally one injection the third and sixth day after said melanoma cell injection, respectively.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Oncology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NO20033492A NO20033492D0 (en) | 2003-08-06 | 2003-08-06 | Use of siRNA in gene silencing to prevent the development of metastases |
US49731403P | 2003-08-25 | 2003-08-25 | |
PCT/NO2004/000238 WO2005040187A2 (en) | 2003-08-06 | 2004-08-05 | The use of sirna silencing in the prevention of metastasis |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1660658A2 true EP1660658A2 (en) | 2006-05-31 |
Family
ID=34525618
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP04816701A Withdrawn EP1660658A2 (en) | 2003-08-06 | 2004-08-05 | The use of sirna silencing in the prevention of metastasis |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1660658A2 (en) |
JP (1) | JP2007501225A (en) |
AU (1) | AU2004284013A1 (en) |
CA (1) | CA2534996A1 (en) |
WO (1) | WO2005040187A2 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7696318B2 (en) | 2005-07-13 | 2010-04-13 | Novo Nordisk Health Care Ag | Host cell protein knock-out cells for production of therapeutic proteins |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE244579T1 (en) * | 1997-01-22 | 2003-07-15 | Univ Texas | TISSUE FACTOR METHODS AND COMPOSITIONS FOR COAGULATION AND TREATMENT OF TUMORS |
WO2001005353A2 (en) * | 1999-07-14 | 2001-01-25 | Novo Nordisk A/S | USE OF FVIIa OR A TISSUE FACTOR ANTAGONIST FOR REGULATING GENE EXPRESSION AND CELL MIGRATION OR CHEMOTAXIS |
CA2475447A1 (en) * | 2002-02-07 | 2003-08-14 | Hans Prydz | Short interfering rna molecules directed towards a tissue factor coding nucleic acid |
US20050096289A1 (en) * | 2002-02-07 | 2005-05-05 | Hans Prydz | Methods and compositions for modulating tissue factor |
-
2004
- 2004-08-05 EP EP04816701A patent/EP1660658A2/en not_active Withdrawn
- 2004-08-05 WO PCT/NO2004/000238 patent/WO2005040187A2/en active Search and Examination
- 2004-08-05 AU AU2004284013A patent/AU2004284013A1/en not_active Abandoned
- 2004-08-05 JP JP2006522519A patent/JP2007501225A/en active Pending
- 2004-08-05 CA CA002534996A patent/CA2534996A1/en not_active Abandoned
Non-Patent Citations (3)
Title |
---|
AMARZGUIOUI M. ET AL: "Ex vivo and in vivo delivery of anti-tissue factor short interfering RNA inhibits mouse pulmonary metastasis of B16 melanoma cells", CLINICAL CANCER RESEARCH, vol. 12, July 2006 (2006-07-01), pages 4055 - 4061 * |
BROMBERG M. ET AL: "Tissue factor promotes melanoma metastasis by a pathway independent of blood coagulation", PROC.NATL.ACAD.SCI., vol. 92, August 1995 (1995-08-01), pages 8205 - 8209 * |
Declaration of Pr. Inge Sandlie of the university of Oslo as filed with the letter of 10.08.2006. * |
Also Published As
Publication number | Publication date |
---|---|
JP2007501225A (en) | 2007-01-25 |
CA2534996A1 (en) | 2005-05-06 |
AU2004284013A1 (en) | 2005-05-06 |
WO2005040187A2 (en) | 2005-05-06 |
WO2005040187A3 (en) | 2005-09-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2019200789B2 (en) | Multimeric oligonucleotide compounds | |
CN111107853B (en) | RNAi agents and compositions for inhibiting expression of apolipoprotein C-III (APOC 3) | |
KR101770435B1 (en) | Treatment of apolipoprotein-a1 related diseases by inhibition of natural antisense transcript to apolipoproteina1 | |
KR101829469B1 (en) | Treatment of erythropoietin (epo) related diseases by inhibition of natural antisense transcript to epo | |
WO2018140920A1 (en) | Compositions and methods for inhibition of factor xii gene expression | |
US20160272970A1 (en) | RNA Interference Agents | |
KR101718297B1 (en) | Organic compositions to treat hsf1-related diseases | |
WO2016149331A2 (en) | Compositions and methods for inhibiting gene expression of factor xii | |
TW201505637A (en) | Complement component C5 iRNA compositions and methods of use thereof | |
US20180282728A1 (en) | Organic compositions to treat beta-catenin-related diseases | |
US20050153918A1 (en) | Methods and compositions relating to hnRNP A1, A1B, A2, and B1 nucleic acid molecules | |
AU2003258183B2 (en) | Use of antisense oligonucleotides to inhibit the expression of Akt-1 | |
JP2007510408A (en) | IAP nucleobase oligomers and nucleobase oligomer complexes and their use | |
US20100273998A1 (en) | Methods and compositions for modulating tissue factor | |
AU2003206267B2 (en) | Short interfering RNA molecules directed towards a tissue factor coding nucleic acid | |
WO2005040187A2 (en) | The use of sirna silencing in the prevention of metastasis | |
EP3237619A1 (en) | Compositions and methods for inhibiting expression of adamts-5 and adam17 | |
WO2023143483A1 (en) | Compositions and methods for inhibiting expression of prekallikrein (pkk) protein | |
WO2024131916A1 (en) | Compositions and methods for inhibiting expression of 17beta-hydroxysteroid dehydrogenase type 13 (hsd17b13) | |
WO2024182580A2 (en) | Rnai agents for inhibiting expression of proprotein convertase subtilisin kexin 9 (pcsk9), pharmaceutical compositions thereof, and methods of use | |
OA19513A (en) | RNAI agents and compositions for inhibiting expression of apolipoprotein C-lll (APOC3). | |
US20120232126A1 (en) | Multi-targets interfering rna molecules and their applications |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20060306 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR |
|
DAX | Request for extension of the european patent (deleted) | ||
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: SIRNASENSE AS |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: AMARZGUIOUI, MOHAMMED Inventor name: PRYDZ, HANS PETER B. Inventor name: HOLEN, TORGEIR |
|
17Q | First examination report despatched |
Effective date: 20090212 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20090825 |