WO2005112636A2 - COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF PRIMATE POLYOMAVIRUS GENES - Google Patents
COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF PRIMATE POLYOMAVIRUS GENES Download PDFInfo
- Publication number
- WO2005112636A2 WO2005112636A2 PCT/US2005/016597 US2005016597W WO2005112636A2 WO 2005112636 A2 WO2005112636 A2 WO 2005112636A2 US 2005016597 W US2005016597 W US 2005016597W WO 2005112636 A2 WO2005112636 A2 WO 2005112636A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sirna
- gene
- jcv
- sense
- rna strand
- Prior art date
Links
- 108020004459 Small interfering RNA Proteins 0.000 title claims abstract description 265
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 82
- 241001505332 Polyomavirus sp. Species 0.000 title claims abstract description 30
- 241000288906 Primates Species 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims description 74
- 239000000203 mixture Substances 0.000 title description 7
- 230000005764 inhibitory process Effects 0.000 title description 6
- 101710134784 Agnoprotein Proteins 0.000 claims abstract description 116
- 101710124584 Probable DNA-binding protein Proteins 0.000 claims abstract description 116
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 101
- 101710128836 Large T antigen Proteins 0.000 claims abstract description 57
- 230000014509 gene expression Effects 0.000 claims abstract description 49
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 32
- 201000010099 disease Diseases 0.000 claims abstract description 29
- 210000004027 cell Anatomy 0.000 claims description 73
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 71
- 239000002773 nucleotide Substances 0.000 claims description 57
- 125000003729 nucleotide group Chemical group 0.000 claims description 54
- 208000015181 infectious disease Diseases 0.000 claims description 44
- 239000002502 liposome Substances 0.000 claims description 36
- 239000013603 viral vector Substances 0.000 claims description 32
- 239000003184 complementary RNA Substances 0.000 claims description 30
- 238000011282 treatment Methods 0.000 claims description 30
- 108020005544 Antisense RNA Proteins 0.000 claims description 27
- 239000013612 plasmid Substances 0.000 claims description 27
- 150000007523 nucleic acids Chemical group 0.000 claims description 23
- 230000015556 catabolic process Effects 0.000 claims description 22
- 238000006731 degradation reaction Methods 0.000 claims description 22
- 239000013598 vector Substances 0.000 claims description 21
- 206010028980 Neoplasm Diseases 0.000 claims description 20
- 230000002401 inhibitory effect Effects 0.000 claims description 19
- 239000008194 pharmaceutical composition Substances 0.000 claims description 16
- 230000010076 replication Effects 0.000 claims description 12
- 201000011510 cancer Diseases 0.000 claims description 10
- 238000001802 infusion Methods 0.000 claims description 10
- 208000016192 Demyelinating disease Diseases 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- 230000014207 opsonization Effects 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 108091026890 Coding region Proteins 0.000 claims description 7
- 101710163270 Nuclease Proteins 0.000 claims description 6
- 238000007792 addition Methods 0.000 claims description 6
- 238000006467 substitution reaction Methods 0.000 claims description 6
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 claims description 5
- 230000004075 alteration Effects 0.000 claims description 5
- 230000001939 inductive effect Effects 0.000 claims description 5
- 238000001990 intravenous administration Methods 0.000 claims description 5
- 208000027580 BK-virus nephropathy Diseases 0.000 claims description 4
- 206010065381 Polyomavirus-associated nephropathy Diseases 0.000 claims description 4
- 241000711975 Vesicular stomatitis virus Species 0.000 claims description 4
- MMXKIWIWQPKTIK-KPRKPIBOSA-N [(2r,3s,5r)-3-hydroxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methyl [(2r,3s,5r)-5-(5-methyl-2,4-dioxopyrimidin-1-yl)-2-(phosphonooxymethyl)oxolan-3-yl] hydrogen phosphate Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(O)=O)[C@@H](O)C1 MMXKIWIWQPKTIK-KPRKPIBOSA-N 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 4
- 208000005017 glioblastoma Diseases 0.000 claims description 4
- 239000003446 ligand Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 102000018697 Membrane Proteins Human genes 0.000 claims description 3
- 108010052285 Membrane Proteins Proteins 0.000 claims description 3
- 206010027406 Mesothelioma Diseases 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 238000012217 deletion Methods 0.000 claims description 3
- 230000037430 deletion Effects 0.000 claims description 3
- 230000008021 deposition Effects 0.000 claims description 3
- 238000000151 deposition Methods 0.000 claims description 3
- 238000001361 intraarterial administration Methods 0.000 claims description 3
- 238000007911 parenteral administration Methods 0.000 claims description 3
- 241001529453 unidentified herpesvirus Species 0.000 claims description 3
- 208000000172 Medulloblastoma Diseases 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 238000007920 subcutaneous administration Methods 0.000 claims description 2
- 238000010254 subcutaneous injection Methods 0.000 claims description 2
- 239000007929 subcutaneous injection Substances 0.000 claims description 2
- 210000005166 vasculature Anatomy 0.000 claims description 2
- 241001115402 Ebolavirus Species 0.000 claims 2
- 241000725171 Mokola lyssavirus Species 0.000 claims 2
- 241000711798 Rabies lyssavirus Species 0.000 claims 2
- 229940124447 delivery agent Drugs 0.000 claims 2
- 230000001177 retroviral effect Effects 0.000 claims 2
- 210000005260 human cell Anatomy 0.000 claims 1
- 230000009368 gene silencing by RNA Effects 0.000 abstract description 22
- 208000001676 Polyomavirus Infections Diseases 0.000 abstract description 8
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 abstract description 8
- XDMCWZFLLGVIID-SXPRBRBTSA-N O-(3-O-D-galactosyl-N-acetyl-beta-D-galactosaminyl)-L-serine Chemical compound CC(=O)N[C@H]1[C@H](OC[C@H]([NH3+])C([O-])=O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 XDMCWZFLLGVIID-SXPRBRBTSA-N 0.000 description 59
- 102000004169 proteins and genes Human genes 0.000 description 34
- 241000829111 Human polyomavirus 1 Species 0.000 description 27
- 230000003612 virological effect Effects 0.000 description 24
- 206010036807 progressive multifocal leukoencephalopathy Diseases 0.000 description 22
- 210000001130 astrocyte Anatomy 0.000 description 20
- 238000004519 manufacturing process Methods 0.000 description 18
- 230000008685 targeting Effects 0.000 description 17
- 108091030071 RNAI Proteins 0.000 description 14
- 108091028043 Nucleic acid sequence Proteins 0.000 description 12
- 230000007423 decrease Effects 0.000 description 12
- 230000000692 anti-sense effect Effects 0.000 description 11
- 239000000427 antigen Substances 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 230000000295 complement effect Effects 0.000 description 11
- 230000001404 mediated effect Effects 0.000 description 11
- 238000001262 western blot Methods 0.000 description 11
- 108091081021 Sense strand Proteins 0.000 description 10
- 108090000565 Capsid Proteins Proteins 0.000 description 9
- 102100023321 Ceruloplasmin Human genes 0.000 description 9
- 108010026056 polyomavirus agnoprotein Proteins 0.000 description 9
- 208000030507 AIDS Diseases 0.000 description 8
- 239000002202 Polyethylene glycol Substances 0.000 description 8
- 239000002299 complementary DNA Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 229920001223 polyethylene glycol Polymers 0.000 description 8
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 7
- 230000001413 cellular effect Effects 0.000 description 7
- 210000003169 central nervous system Anatomy 0.000 description 7
- 239000000284 extract Substances 0.000 description 7
- 230000001605 fetal effect Effects 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 6
- 210000004748 cultured cell Anatomy 0.000 description 6
- 229920001451 polypropylene glycol Polymers 0.000 description 6
- 238000013518 transcription Methods 0.000 description 6
- 230000035897 transcription Effects 0.000 description 6
- 241000701161 unidentified adenovirus Species 0.000 description 6
- 230000006648 viral gene expression Effects 0.000 description 6
- 230000009385 viral infection Effects 0.000 description 6
- 239000013607 AAV vector Substances 0.000 description 5
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 108010067390 Viral Proteins Proteins 0.000 description 5
- 210000000234 capsid Anatomy 0.000 description 5
- 238000011278 co-treatment Methods 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 5
- 230000001629 suppression Effects 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 230000029812 viral genome replication Effects 0.000 description 5
- 241000701022 Cytomegalovirus Species 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 108091028664 Ribonucleotide Proteins 0.000 description 4
- 108010052104 Viral Regulatory and Accessory Proteins Proteins 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 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 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 102000034356 gene-regulatory proteins Human genes 0.000 description 4
- 108091006104 gene-regulatory proteins Proteins 0.000 description 4
- 239000007943 implant Substances 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 239000000546 pharmaceutical excipient Substances 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 229920001282 polysaccharide Polymers 0.000 description 4
- 239000005017 polysaccharide Substances 0.000 description 4
- 150000004804 polysaccharides Chemical class 0.000 description 4
- 239000002336 ribonucleotide Substances 0.000 description 4
- 125000002652 ribonucleotide group Chemical group 0.000 description 4
- 238000010561 standard procedure Methods 0.000 description 4
- 238000003146 transient transfection Methods 0.000 description 4
- 230000014616 translation Effects 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 108020004394 Complementary RNA Proteins 0.000 description 3
- 230000004543 DNA replication Effects 0.000 description 3
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 3
- 101710121996 Hexon protein p72 Proteins 0.000 description 3
- 101710125418 Major capsid protein Proteins 0.000 description 3
- 206010029113 Neovascularisation Diseases 0.000 description 3
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Natural products N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 3
- 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 3
- 208000036142 Viral infection Diseases 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 201000007455 central nervous system cancer Diseases 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000002596 correlated effect Effects 0.000 description 3
- 239000005547 deoxyribonucleotide Substances 0.000 description 3
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000003365 immunocytochemistry Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000006722 reduction reaction Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- -1 ribonucleoside phosphoramidites Chemical class 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 229960000303 topotecan Drugs 0.000 description 3
- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 2
- IQFYYKKMVGJFEH-BIIVOSGPSA-N 2'-deoxythymidine Natural products O=C1NC(=O)C(C)=CN1[C@@H]1O[C@@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-BIIVOSGPSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical class [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 2
- 206010012305 Demyelination Diseases 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 2
- 102100023387 Endoribonuclease Dicer Human genes 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101000907904 Homo sapiens Endoribonuclease Dicer Proteins 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 102100034349 Integrase Human genes 0.000 description 2
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 2
- 241000713666 Lentivirus Species 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 208000012902 Nervous system disease Diseases 0.000 description 2
- 238000000636 Northern blotting Methods 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 108010039918 Polylysine Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 101100108356 Squirrel monkey polyomavirus agnogene gene Proteins 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 241000906446 Theraps Species 0.000 description 2
- 108091036066 Three prime untranslated region Proteins 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000003140 astrocytic effect Effects 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 208000025997 central nervous system neoplasm Diseases 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 150000002270 gangliosides Chemical class 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- 229920002674 hyaluronan Polymers 0.000 description 2
- 229960003160 hyaluronic acid Drugs 0.000 description 2
- 229920001477 hydrophilic polymer Polymers 0.000 description 2
- 230000002895 hyperchromatic effect Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 230000002101 lytic effect Effects 0.000 description 2
- 229920001427 mPEG Polymers 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 2
- 210000004498 neuroglial cell Anatomy 0.000 description 2
- 230000002276 neurotropic effect Effects 0.000 description 2
- 210000004248 oligodendroglia Anatomy 0.000 description 2
- 229920001542 oligosaccharide Chemical class 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 229940124531 pharmaceutical excipient Drugs 0.000 description 2
- 229920000962 poly(amidoamine) Polymers 0.000 description 2
- 229920000656 polylysine Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000002213 purine nucleotide Substances 0.000 description 2
- 150000003212 purines Chemical class 0.000 description 2
- 238000001959 radiotherapy Methods 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000004055 small Interfering RNA Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108050006400 Cyclin Proteins 0.000 description 1
- 102000016736 Cyclin Human genes 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-VANFPWTGSA-N D-mannopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H]1O AEMOLEFTQBMNLQ-VANFPWTGSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 201000011001 Ebola Hemorrhagic Fever Diseases 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- 208000031448 Genomic Instability Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010048748 Graft loss Diseases 0.000 description 1
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical class C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 241000701460 JC polyomavirus Species 0.000 description 1
- 206010023163 JC virus infection Diseases 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
- 208000030289 Lymphoproliferative disease Diseases 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000714177 Murine leukemia virus Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000014767 Myeloproliferative disease Diseases 0.000 description 1
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- REYJJPSVUYRZGE-UHFFFAOYSA-N Octadecylamine Chemical compound CCCCCCCCCCCCCCCCCCN REYJJPSVUYRZGE-UHFFFAOYSA-N 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229920002230 Pectic acid Polymers 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 229920002873 Polyethylenimine Polymers 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 102000000574 RNA-Induced Silencing Complex Human genes 0.000 description 1
- 108010016790 RNA-Induced Silencing Complex Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 206010037742 Rabies Diseases 0.000 description 1
- 208000035977 Rare disease Diseases 0.000 description 1
- WINXNKPZLFISPD-UHFFFAOYSA-M Saccharin sodium Chemical compound [Na+].C1=CC=C2C(=O)[N-]S(=O)(=O)C2=C1 WINXNKPZLFISPD-UHFFFAOYSA-M 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 101710183280 Topoisomerase Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- IAJILQKETJEXLJ-RSJOWCBRSA-N aldehydo-D-galacturonic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-RSJOWCBRSA-N 0.000 description 1
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- VSRXQHXAPYXROS-UHFFFAOYSA-N azanide;cyclobutane-1,1-dicarboxylic acid;platinum(2+) Chemical compound [NH2-].[NH2-].[Pt+2].OC(=O)C1(C(O)=O)CCC1 VSRXQHXAPYXROS-UHFFFAOYSA-N 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 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
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 229960005069 calcium Drugs 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011692 calcium ascorbate Substances 0.000 description 1
- 235000010376 calcium ascorbate Nutrition 0.000 description 1
- 229940047036 calcium ascorbate Drugs 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229960002713 calcium chloride Drugs 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 239000004227 calcium gluconate Substances 0.000 description 1
- 229960004494 calcium gluconate Drugs 0.000 description 1
- 235000013927 calcium gluconate Nutrition 0.000 description 1
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 description 1
- 239000001527 calcium lactate Substances 0.000 description 1
- 229960002401 calcium lactate Drugs 0.000 description 1
- 235000011086 calcium lactate Nutrition 0.000 description 1
- BLORRZQTHNGFTI-ZZMNMWMASA-L calcium-L-ascorbate Chemical compound [Ca+2].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] BLORRZQTHNGFTI-ZZMNMWMASA-L 0.000 description 1
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 description 1
- BHRQIJRLOVHRKH-UHFFFAOYSA-L calcium;2-[bis[2-[bis(carboxylatomethyl)amino]ethyl]amino]acetate;hydron Chemical compound [Ca+2].OC(=O)CN(CC(O)=O)CCN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O BHRQIJRLOVHRKH-UHFFFAOYSA-L 0.000 description 1
- 150000004653 carbonic acids Chemical class 0.000 description 1
- 229960004562 carboplatin Drugs 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 239000003534 dna topoisomerase inhibitor Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 229960002949 fluorouracil Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 210000004884 grey matter Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002687 intercalation Effects 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- BQINXKOTJQCISL-GRCPKETISA-N keto-neuraminic acid Chemical compound OC(=O)C(=O)C[C@H](O)[C@@H](N)[C@@H](O)[C@H](O)[C@H](O)CO BQINXKOTJQCISL-GRCPKETISA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 230000001589 lymphoproliferative effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 238000007479 molecular analysis Methods 0.000 description 1
- 201000011682 nervous system cancer Diseases 0.000 description 1
- 210000003061 neural cell Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- CERZMXAJYMMUDR-UHFFFAOYSA-N neuraminic acid Natural products NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO CERZMXAJYMMUDR-UHFFFAOYSA-N 0.000 description 1
- 201000001119 neuropathy Diseases 0.000 description 1
- 230000007823 neuropathy Effects 0.000 description 1
- 231100000189 neurotoxic Toxicity 0.000 description 1
- 230000002887 neurotoxic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- LCLHHZYHLXDRQG-ZNKJPWOQSA-N pectic acid Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)O[C@H](C(O)=O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](OC2[C@@H]([C@@H](O)[C@@H](O)[C@H](O2)C(O)=O)O)[C@@H](C(O)=O)O1 LCLHHZYHLXDRQG-ZNKJPWOQSA-N 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 208000033808 peripheral neuropathy Diseases 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical group 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 229920000191 poly(N-vinyl pyrrolidone) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000010318 polygalacturonic acid Substances 0.000 description 1
- 229920000155 polyglutamine Polymers 0.000 description 1
- 108010040003 polyglutamine Proteins 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
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 125000000561 purinyl group Chemical group N1=C(N=C2N=CNC2=C1)* 0.000 description 1
- 239000002719 pyrimidine nucleotide Substances 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 238000006268 reductive amination reaction Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 239000011877 solvent mixture Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 230000002739 subcortical effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 229940044693 topoisomerase inhibitor Drugs 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 229940073585 tromethamine hydrochloride Drugs 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 229920003169 water-soluble polymer Polymers 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 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
- C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- 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
- C12N2310/50—Physical structure
- C12N2310/53—Physical structure partially self-complementary or closed
Definitions
- This invention relates to the regulation of primate polyomavirus gene expression, such as human neurotropic polyomavirus JCV gene expression, by small interfering RNA.
- primate polyomavirus infection and diseases associated with primate polyomavirus infection can be treated.
- PML Progressive multifocal leukoencephalopathy
- CSF cerebrospinal fluid
- PML histological hallmarks of PML include multifocal demyelinated lesions with enlarged hyperchromatic nuclei in oligodendrocytes and enlarged playful astrocytes with lobulated hyperchromatic nuclei within white matter tracts of the brain (Walker and Padgett, 1983; Cinque et al, 2003). Although in some instances, atypical features that include a unifocal pattern of demyelination and involvement of the gray matter have been reported (Sweeney et al., 1994; for review see Cinque et al., 2003 JNV).
- JCV is a small DNA virus whose genome can be divided into three regions that encompass the transcription control region, the genes responsible for the expression of the viral early protein, T-antigen, and the viral late proteins, VP1, VP2, and VP3.
- the late genome is also responsible for production of the viral auxiliary protein, Agnoprotein.
- T-antigen expression is pivotal for initiation of the viral lytic cycle, as this protein stimulates transcription of the late genes, and induces the process of viral DNA replication (Frisque and White, 1992).
- Recent studies have ascribed an important role for Agnoprotein in transcription and replication of JCV, as inhibition of its production significantly reduces viral gene expression and replication (Safak et al, unpublished observations).
- agnoprotein dysregulates the cell cycle by altering the expression of several cyclins and their associated kinases (Darbinyan et al, 2002).
- JCV infection has been linked to various tumors of central nervous system (CNS) origin, including medullablastoma, glioblastoma, and others. Del Valle L et al., (2001a), supra; Khaiili K (1999), supra. Examination of T-antigen expression in the CNS tumor tissue revealed that not all tumor cells express T-antigen. Evaluation of these tumors for other viral proteins showed a substantial level of agnoprotein in tumors containing the JCV genome. DeValle L et al. (2002), J. Nat. Cancer Inst. 94(4): 267-273.
- RNA interference is a method of post- transcriptional gene regulation that is conserved throughout many eukaryotic organisms. RNAi is induced by short (i.e., ⁇ 30 nucleotide) double stranded RNA (“dsRNA”) molecules which are present in the cell (Fire A et al. (1998), Nature 391_: 806-811). These short dsRNA molecules, called “short interfering RNA” or “siRNA,” cause the destruction of messenger RNAs ("mRNAs”) which share sequence homology with the siRNA to within one nucleotide resolution (Elbashir SM et al. (2001), Genes Dev, 15: 188-200).
- mRNAs messenger RNAs
- siRNA-induced silencing complex or "RISC”
- RISC RNA-induced silencing complex
- the siRNA is apparently recycled much like a multiple-turnover enzyme, with 1 siRNA molecule capable of inducing cleavage of approximately 1000 mRNA molecules.
- siRNA-mediated RNAi degradation of an mRNA is therefore more effective than currently available technologies for inhibiting expression of a target gene.
- Elbashir SM et al. (2001), supra has shown that synthetic siRNA of 21 and 22 nucleotides in length, and which have short 3' overhangs, are able to induce RNAi of target mRNA in a Drosophila cell lysate.
- RNAi degradation with synthetic siRNA (Elbashir SM et al. (2001) Nature, 4 A : 494-498), and RNAi degradation induced by synthetic siRNA has recently been shown in living mice (McCaffrey AP et al. (2002), Nature, 418: 38-39; Xia H et al. (2002), Nat. Biotech. 20: 1006-1010).
- the therapeutic potential of siR ⁇ A-induced R ⁇ Ai degradation has been demonstrated in several recent in vitro studies, including the siR ⁇ A-directed inhibition of HIV-1 infection ( ⁇ ovina CD et al. (2002), Nat. Med. 8: 681-686) and reduction of neurotoxic polyglutamine disease protein expression (Xia H et al. (2002), supra).
- the present invention is directed to siR ⁇ A which specifically target and cause R ⁇ Ai-induced degradation of mR ⁇ A from primate polyomavirus genes, in particular from the agnoprotein and large T antigen genes of JCV. These siR ⁇ A degrade agnoprotein and large T antigen mR ⁇ A in substoichiometric amounts.
- the siR ⁇ A compounds and compositions of the invention can be used to inhibit JCV, BKV and/or SV40 infection and replication, and treat diseases associated with JCV, BKV and/or SV40 infection.
- the siR ⁇ A of the invention are useful for treating cancer or progressive multifocal leukoencephalopathy ("PLM").
- the invention provides an isolated siR ⁇ A which targets JCV agnoprotein gene or large T antigen gene mR ⁇ A, or an alternative splice form or mutant thereof.
- the siR ⁇ A comprises a sense R ⁇ A strand and an antisense R ⁇ A strand which form an R ⁇ A duplex.
- the sense R ⁇ A strand comprises a nucleotide sequence substantially identical to a target sequence of about 19 to about 25 contiguous nucleotides in the target mRNA.
- the mRNA produced from BKV and/or SV40 genes can have sequences in common with JCV mRNA; thus, the siRNA of the invention that target and degrade JCV mRNA also target and degrade BKV and/or SV40 mRNA, when the BKV and/or SV40 mRNA contains a target sequence in common with the JCV mRNA.
- the invention also provides recombinant plasmids and viral vectors which express the siRNA of the invention, as well as pharmaceutical compositions comprising the siRNA of the invention and a pharmaceutically acceptable carrier.
- the invention further provides a method of inhibiting expression of JCV agnoprotein gene and/or large T antigen gene mRNA, or alternative splice forms or mutants thereof, comprising administering to a subject an effective amount of one or more of the siRNA of the invention such that the target mRNA is degraded.
- the invention further provides a method of inhibiting expression of JCV VPl protein, comprising administering to a subject an effective amount of one or more of the siRNA targeted to JCV agnoprotein gene and/or large T antigen gene mRNA, or alternative splice forms or mutants thereof.
- the invention further provides a method of inhibiting JCV infection or replication in a subject, comprising administering to a subject an effective amount of an siRNA targeted to JCV agnoprotein gene and/or large T antigen gene mRNA, or alternative splice forms or mutants thereof.
- the invention further provides a method of treating diseases associated with JCV infection, for example cancer or PML, comprising administering to a subject in need of such treatment an effective amount of an siRNA targeted to JCV agnoprotein gene and/or large T antigen gene mRNA, or alternative splice forms or mutants thereof.
- diseases associated with JCV infection for example cancer or PML
- administering comprising administering to a subject in need of such treatment an effective amount of an siRNA targeted to JCV agnoprotein gene and/or large T antigen gene mRNA, or alternative splice forms or mutants thereof.
- JCV T-antigen siRNA decreases expression of JCV proteins in transiently transfected and infected primary human astrocytes.
- Primary human fetal astrocytes were prepared as described previously and were seeded onto 6 well plates at a density of 500,000 cells/well (Radhakrishnan et al, 2003).
- FuGENE 6 For transient transfections, cells were transfected using FuGENE 6 with plasmid expressing JCV T-antigen (Kerr et al, 1993).
- the cells were transfected with double-stranded 21-bp siRNA for JCV T-antigen targeting nt 4256-4276 of the Mad-1 isolate of JCV, sense strand 5'- AAGUCUUUAGGGUCUUCUACCUdTdT-3'.
- the siRNAs were prepared as double stranded, 2'-deprotected and desalted oligonucleotide and were utilized according to the manufacturer's directions (Dharmacon).
- 100 pmol of siRNA was mixed with 3 ul of Oligofectamine (Invitrogen), diluted in OptiMEM (Invitrogen) and incubated with the cell cultures for 4 h at 37°C in serum- and antibiotic-free conditions.
- Activity is presented as a difference in fold change with the background activity of the JCV late promoter arbitrarily set a 1 from 4 experiments. Standard deviations are indicated by error bars.
- C Primary astrocytes were infected with JCV Mad-4 strain at an M.O.I, of 1 in serum-free media for 3 h at 37°C. Uninfected and infected cells were then transfected with T-antigen siRNA at days 1, 5, and 10 post-infection and were harvested at day 15.
- FIG. 1 JCV Agnoprotein siRNA decreases Agnogene expression as well as other viral proteins in primary human astrocytes.
- Primary human fetal astrocyte preparation, transient transfections, siRNA treatment, and Western blotting were performed as described in the text and the legend to Figure 1.
- Cells were transfected with a plasmid containing the JCV Agnoprotein fused to YFP (Darbinyan et al, 2002).
- JCV Agnoprotein siRNA targeted nt 324-342 of the Mad-1 isolate of JCV, sense strand 5'- AACCUGGAGUGGAACUAAAdTdT-3' while non-specific siRNA (ns siRNA) targeted nt 435-453 of the Dunlop strain of BKV, sense strand 5'- AACCUGGACUGGAACAAAAdTdT-3'.
- ns siRNA non-specific siRNA
- the two base pair mismatches between JCV and BKV Agnoprotein sequences are underlined.
- A. Whole cell extracts prepared from transfected astrocytes 24 h after treatment with specific or non-specific siRNAs were analyzed by Western blotting for the presence of Agnoprotein and the unrelated cellular factor, grb-2 (upper and lower panels, respectively).
- Agnoprotein abrogate their expression in primary human astrocytes as well as of the JCV late protein, VPl, in infected cells.
- Primary human fetal astrocyte preparation, transient transfections, siRNA treatment, and Western blotting were performed as described in the text and the legend to Figure 1.
- A. Whole cell extracts from astrocytes transfected with expression plasmids for JCV T- antigen, YFP-Agnoprotein, or both were prepared from the cultures 24 h after siRNA treatment and were analyzed by Western blotting for the presence of T- antigen and Agnoprotein as well as for the unrelated grb-2.
- Astrocyte cultures were infected with the JCV Mad-4 strain of JCV and were then transfected with siRNA targeting JCV T-antigen, Agnoprotein, or both at days 1, 5, and 10 post-infection.
- Western blotting was performed on whole cell extracts harvested at day 15 post-infection for the presence of JCV T-antigen and Agnoprotein, as well as the viral late protein, VP 1, and the cellular protein, grb-2.
- FIG. 4 Immunocytochemistry of primary astrocytes infected with JCV reveals alterations in JCV protein levels upon JCV T-antigen and Agnoprotein siRNA treatment.
- Cells were infected with JCV and treated at days 1, 5, and 10 post-infection with JCV T-antigen siRNA, JCV Agnoprotein siRNA, or both. Cells were also treated with BKV siRNA. Cells were subcultured and plated onto poly-L-lysine coated chamber slides (Falcon) on day 13 and were fixed on day 15 post-infection with ice-cold acetone for 3 min.
- Viral proteins were detected by immunocytochemistry as described previously (Radhakrishnan et al, 2003) using the same primary antibodies as for Western blot analysis (see legend to Figure 1). Proteins were visualized with fluorescein-conjugated secondary antibodies.
- Figure 5 shows the alignment of nucleic acid sequences from JCV, BK virus (BKV) and SV40 agnoprotein genes. Identical bases are shown in grey; non-identical bases are shown in white.
- Figures 6a to 6g show the alignment of nucleic acid sequences from JCV, BKV and SV40 large T antigen genes. Identical bases are shown in grey; non-identical bases are shown in white.
- nucleic acid sequences herein are given in the 5' to 3' direction. Also, all deoxyribonucleotides in a nucleic acid sequence are represented by capital letters (e.g., deoxythymidine is "T"), and ribonucleotides in a nucleic acid sequence are represented by lower case letters (e.g., uridine is "u").
- the human polyomavirus, JCV encodes two regulatory proteins at the early (T-antigen) and the late (agnoprotein) phase of viral infection whose activities are important for the production of the viral capsid proteins and dysregulation of several host factors and their functions.
- T-antigen early
- agnoprotein late
- siRNAs target expression of T- antigen and Agnoprotein in human astrocytic cells.
- Treatment of the cells with specific siRNA oligonucleotides targeting a conserved region of T-antigen, nt 4256 to 4276 (Mad-1 strain) caused greater than 50% decline in the level of T- antigen and its transcriptional activity upon the viral capsid genes.
- compositions and methods comprising siRNA targeted to JCV agnoprotein gene or large T antigen gene mRNA are advantageously used to inhibit JCV infection and replication, in particular for the treatment of JCV- associated diseases.
- the siRNA of the invention are believed to cause the RNAi-mediated degradation of these mRNAs, so that the protein products of the JCV agnoprotein gene or large T antigen gene are not produced or are produced in reduced amounts. Because JCV agnoprotein gene and the large T antigen gene are required for the JCV life-cycle, the siRNA-mediated degradation of JCV agnoprotein gene or large T antigen gene mRNA inhibits JCV infection and thus any pathology related to such infection; for example JCV-related demyelinating disease.
- siRNA which is "targeted to the JCV agnoprotein gene or large T antigen gene mRNA” means siRNA in which a first strand of the duplex is substantially identical to the nucleotide sequence of a portion of the JCV agnoprotein gene or large T antigen gene mRNA sequence. It is understood that the second strand of the siRNA duplex is complementary to both the first strand of the siRNA duplex and to the same portion of the JCV agnoprotein gene or large T antigen gene mRNA.
- the invention therefore provides isolated siRNA comprising short double-stranded RNA from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 to about 25 nucleotides in length, that are targeted to the target mRNA.
- the siRNA's comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson- Crick base-pairing interactions (hereinafter "base-paired").
- base-paired standard Watson- Crick base-pairing interactions
- the sense strand comprises a nucleic acid sequence which is substantially identical to a target sequence contained within the target mRNA.
- nucleic acid sequence "substantially identical" to a target sequence contained within the target mRNA is a nucleic acid sequence which is identical to the target sequence, or which differs from the target sequence by one or more nucleotides.
- Sense strands of the invention which comprise nucleic acid sequences substantially identical to a target sequence are characterized in that siRNA comprising such sense strands induce RNAi- mediated degradation of mRNA containing the target sequence.
- an siRNA of the invention can comprise a sense strand which comprises nucleic acid sequences that differ from a target sequence by one, two or three or more nucleotides, as long as RNAi-mediated degradation of the target mRNA is induced by the siRNA.
- the sense and antisense strands of the present siRNA can comprise two complementary, single-stranded RNA molecules or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded "hairpin" area.
- hairpin area of the latter type of siRNA molecule is cleaved intracellularly by the "Dicer" protein (or its equivalent) to form a siRNA of two individual base-paired RNA molecules (see Tuschl, T. (2002), supra).
- the siRNA can also contain alterations, substitutions or modifications of one or more ribonucleotide bases.
- the present siRNA can be altered, substituted or modified to contain one or more deoxyribonucleotide bases.
- isolated means synthetic, or altered or removed from the natural state through human intervention.
- an siRNA naturally present in a living animal is not “isolated,” but a synthetic siRNA, or an siRNA partially or completely separated from the coexisting materials of its natural state is “isolated.”
- An isolated siRNA can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the siRNA has been delivered.
- siRNA which are produced inside a cell by natural processes, but which are produced from an "isolated” precursor molecule are themselves “isolated” molecules.
- an isolated dsRNA can be introduced into a target cell, where it is processed by the Dicer protein (or its equivalent) into isolated siRNA.
- target mRNA means JCV agnoprotein gene or large T antigen gene mRNA, or mutant or alternative splice forms of JCV agnoprotein gene or large T antigen gene mRNA. It is understood that genes from other primate polyomaviruses (such as BKV or SV40) can contain nucleotide sequences in common with JCV genes. Thus, mRNA produced from these other primate polyomavirus genes, which contain nucleotide sequences in common with JCV mRNA (see e.g., . Figs. 5 and 6), are considered “target mRNA” for purposes of the present invention.
- a "primate polyomavirus” is any polyomavirus which infects a primate, in particular a human, and includes JCV, BKV and SV40.
- Agnoprotein gene mRNA and large T antigen gene mRNA from any species and strain of primate polyomavirus can be used in the practice of the present invention.
- Preferred agnoprotein gene and large T antigen gene mRNA are those derived from JCV.
- the nucleic acid sequence encoding the JCV agnoprotein gene mRNA is given in SEQ ID NO: 1 as the cDNA equivalent.
- Other JCV agnoprotein gene mRNAs known in the art include those disclosed (as the cDNA equivalents) in GenBank records Accession No. AF187236 (SEQ ID NO: 2), Accession No. AF281599 (SEQ ID NO: 3), Accession No. AF 187234 (SEQ ID NO: 4), Accession No. AF295737 (SEQ ID NO: 5) and Accession No. AF295739 (SEQ ID NO: 6), the entire disclosures of which are herein in incorporated by reference.
- Agnoprotein from human BK polyomavirus (BKV) strains, or from SV40 polyomavirus strains, are also known.
- BKV agnoproteins gene mRNAs are given (as the cDNA equivalents) in GenBank record Accession Nos. M23122 (SEQ ID NO: 7) and D00678 (SEQ ID NO: 8), the disclosures of which are herein incorporated by reference.
- An example of SV40 agnoprotein is given (as the cDNA sequence) in GenBank record Accession No. M99359 (SEQ ID NO: 9), the disclosure of which is herein incorporated by reference.
- the mRNA transcribed from the JCV agnoprotein and large T antigen genes can also be analyzed for alternative splice forms using techniques well-known in the art. Such techniques include reverse transcription- polymerase chain reaction (RT-PCR), northern blotting and in-situ hybridization. Techniques for analyzing mRNA sequences are described, for example, in Busting SA (2000), J. Mol. Endocrinol. 25: 169-193, the entire disclosure of which is herein incorporated by reference. Representative techniques for identifying alternatively spliced mRNAs are also described below.
- databases that contain nucleotide sequences related to a given disease gene can be used to identify alternatively spliced mRNA.
- databases include GenBank and Embase.
- An mRNA or gene sequence from the JCV agnoprotein and large T antigen genes can be used to query such a database to determine whether ESTs representing alternatively spliced mRNAs have been found.
- a technique called "RNAse protection" can also be used to identify alternatively spliced JCV agnoprotein and large T antigen gene mRNAs.
- RNAse protection involves translation of a gene sequence into synthetic RNA, which is hybridized to RNA derived from other cells; for example, cells which are induced to express agnoprotein and large T antigen. The hybridized RNA is then incubated with enzymes that recognize RNA:RNA hybrid mismatches. Smaller than expected fragments indicate the presence of alternatively spliced mRNAs.
- the putative alternatively spliced mRNAs can be cloned and sequenced by methods well Icnown to those skilled in the art.
- RT-PCR can also be used to identify alternatively spliced JCV agnoprotein and large T antigen gene mRNAs.
- mRNA from cells known or induced to express JCV agnoprotein and large T antigen is converted into cDNA by the enzyme reverse transcriptase, using methods within the skill in the art.
- the entire coding sequence of the cDNA is then amplified via PCR using a forward primer located in the 3' untranslated region, and a reverse primer located in the 5' untranslated region.
- the amplified products can be analyzed for alternative splice forms, for example by comparing the size of the amplified products with the size of the expected product from normally spliced mRNA, e.g., by agarose gel electrophoresis. Any change in the size of the amplified product can indicate alternative splicing.
- mutant JCV agnoprotein and large T antigen genes can also be readily identified with the techniques described above for identifying JCV agnoprotein and large T antigen gene mRNA alternative splice forms.
- mutant JCV agnoprotein and large T antigen genes or mRNA include JCV agnoprotein and large T antigen genes or mRNA which differ in sequence from the JCV agnoprotein and large T antigen gene sequences set forth herein.
- allelic forms of the JCV agnoprotein and large T antigen genes, and the mRNA produced from them are considered “mutants" for purposes of this invention.
- JCV agnoprotein gene and large T antigen gene mRNA may contain target sequences in common with its respective alternative splice forms or mutants, or in common with analogous genes from different strains or species of primate polyomaviruses.
- a single siRNA comprising such a common targeting sequence can therefore induce RNAi-mediated degradation of those different mRNAs which contain the common targeting sequence.
- siRNA of the invention which are targeted to JCV gene sequences, also target BKV and SV40 gene sequences.
- An siRNA of the invention that targets BKV sequences can be used to treat polyomavirus associated nephropathy (PVN), which occurs in kidney transplant recipients. PVN is a leading cause of graft loss and there are currently no treatments.
- An siRNA of the invention that targets SV40 gene sequences can be used to treat SV40-induced cancers, including mesothelioma.
- nucleic acid sequence alignments of the JCV, BKV and SV40 agnoprotein genes which indicate the regions of base sequence identity and non-identity, are shown in Fig. 5.
- nucleic acid sequence alignment of JCV, BKV and SV40 large T antigen genes which indicate the regions of base sequence identity and non-identity, are shown in Fig. 6.
- the siRNA of the invention can comprise partially purified RNA, substantially pure RNA, synthetic RNA, or 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.
- Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA; modifications that make the siRNA resistant to nuclease digestion (e.g., the use of 2'- substituted ribonucleotides or modifications to the sugar-phosphate backbone); or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides.
- siRNA which are exposed to serum, lachrymal fluid or other nuclease-rich environments, or which are delivered topically (e.g., by eyedropper), are preferably altered to increase their resistance to nuclease degradation.
- siRNA which are administered intravascularly or topically to the eye can comprise one or more phosphorothioate linkages.
- One or both strands of the siRNA of the invention can also comprise a 3' overhang.
- a "3' overhang” refers to at least one unpaired nucleotide extending from the 3'-end of an RNA strand.
- the siRNA of the invention comprises at least one 3' overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxynucleotides) in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length.
- each strand of the siRNA of the invention can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").
- TT dithymidylic acid
- uu diuridylic acid
- the 3' overhangs can be also stabilized against degradation.
- the overhangs are stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
- substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine nucleotides in the 3' overhangs with 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNAi degradation.
- the absence of a 2'-hydroxyl in the 2'- deoxythymidine significantly enhances the nuclease resistance of the 3' overhang in tissue culture medium.
- the siRNA of the invention comprises the sequence AA(N19)TT or NA(N21), where N is any nucleotide. These siRNA comprise approximately 30-70% GC, and preferably comprise approximately 50% G/C.
- the sequence of the sense siRNA strand corresponds to (N19)TT or N21 (i.e., positions 3 to 23), respectively. In the latter case, the 3' end of the sense siRNA is converted to TT.
- the rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense strand 3' overhangs.
- the antisense RNA strand is then synthesized as the complement to positions 1 to 21 of the sense strand.
- the 3'-most nucleotide residue of the antisense strand can be chosen deliberately.
- the penultimate nucleotide of the antisense strand (complementary to position 2 of the 23-nucleotide sense strand in either embodiment) is generally complementary to the targeted sequence.
- the siRNA of the invention comprises the sequence NAR(N17)YNN, where R is a purine (e.g., A or G) and Y is a pyrimidine (e.g., C or u/T).
- R is a purine
- Y is a pyrimidine
- the respective 21 -nucleotide sense and antisense RNA strands of this embodiment therefore generally begin with a purine nucleotide.
- Such siRNA can be expressed from pol III expression vectors without a change in targeting site, as expression of RNAs from pol III promoters is only believed to be efficient when the first transcribed nucleotide is a purine.
- the siRNA of the invention can be targeted to any stretch of approximately 19-25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence”).
- target sequence any of the target mRNA sequences.
- Techniques for selecting target sequences for siRNA's are given, for example, in Tuschl T et al., "The siRNA User Guide,” revised Oct. 11, 2002, the entire disclosure of which is herein incorporated by reference. "The siRNA User Guide” is available on the world wide web at a website maintained by Dr.
- the sense strand of the present siRNA comprises a nucleotide sequence substantially identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA.
- a target sequence on the target mRNA can be selected from a given cDNA sequence corresponding to the target mRNA, preferably beginning 50 to 100 nt downstream (i.e., in the 3' direction) from the start codon.
- the target sequence can, however, be located in the 5 ' or 3' untranslated regions, or in the region nearby the start codon. Suitable target sequences are given in Table 1. Table 1 also shows the dinucleotides AA and TT, which are located, respectively, at the 5'- and 3 '-most ends of the target sequences.
- the target sequences and the target strands in Table 1 comprise the sense strand of the preferred siRNAs of the invention.
- target sequences given in Table 1 are with reference to the agnoprotein gene or large T antigen gene cDNA, and thus these sequences contain deoxythymidines represented by "T.”
- T deoxythymidines
- uridines uridines
- the siRNA of the invention can be obtained using a number of techniques known to those of skill in the art.
- the siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. published application 2002/0086356 of Tuschl et al., the entire disclosure of which is herein incorporated by reference.
- the siRNA of the invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
- the siRNA can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
- Commercial suppliers of synthetic RNA molecules or synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA) and Cruachem (Glasgow, UK).
- siRNA can also be expressed from recombinant circular or linear DNA plasmids ' using any suitable promoter.
- suitable promoters for expressing siRNA of the invention from a plasmid include, for example, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
- the recombinant plasmids of the invention can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment.
- siRNA expressed from recombinant plasmids can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly. The use of recombinant plasmids to deliver siRNA of the invention to cells in vivo is discussed in more detail below. [0058] siRNA of the invention can be expressed from a recombinant plasmid either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
- plasmids suitable for expressing siRNA of the invention are within the skill in the art. See, for example Tuschl, T. (2002), Nat. Biotechnol, 20: 446-448; Brummelkamp TR et al. (2002), Science 296- 550- 553; Miyagishi M et al. (2002), Nat. Biotechnol. 20: 497-500; Paddison PJ et al. (2002), Genes Dev. 16: 948-958; Lee NS et al. (2002), Nat. Biotechnol. 20: 500-505; and Paul CP et al. (2002), Nat. Biotechnol. 20: 505-508, the entire disclosures of which are herein incorporated by reference.
- a plasmid expressing an siR ⁇ A of the invention comprises a sense R ⁇ A strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 R ⁇ A promoter, and an antisense R ⁇ A strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 R ⁇ A promoter.
- a plasmid can be used in producing an recombinant adeno-associated viral vector for expressing an siR ⁇ A of the invention.
- in operable connection with a polyT termination sequence means that the nucleic acid sequences encoding the sense or antisense strands are immediately adjacent to the polyT termination signal in the 5' direction. During transcription of the sense or antisense sequences from the plasmid, the polyT termination signals act to terminate transcription.
- promoter under the control of a promoter means that the nucleic acid sequences encoding the sense or antisense strands are located 3' of the promoter, so that the promoter can initiate transcription of the sense or antisense coding sequences.
- the siR ⁇ A of the invention can also be expressed from recombinant viral vectors intracellularly in vivo.
- the recombinant viral vectors of the invention comprise sequences encoding the siRNA of the invention and any suitable promoter for expressing the siRNA sequences. Suitable promoters include, for example, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
- the recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment. The use of recombinant viral vectors to deliver siRNA of the invention to cells in vivo is discussed in more detail below.
- siRNA of the invention can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
- Any viral vector capable of accepting the coding sequences for the siRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g, lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.
- AV adenovirus
- AAV adeno-associated virus
- retroviruses e.g, lentiviruses (LV), Rhabdoviruses, murine leukemia virus
- herpes virus and the like.
- the tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
- lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
- AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes.
- an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2.
- This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.
- AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
- Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the siRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis MA (1988), Biotechniques 6: 608-614; Miller AD (1990), Hum Gene Therap. I: 5-14; Anderson WF (1998), Nature 392: 25- 30; and Rubinson DA et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.
- Preferred viral vectors are those derived from AV and AAV.
- the siRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or HI RNA promoters, or the cytomegalovirus (CMV) promoter.
- CMV cytomegalovirus
- a suitable AV vector for expressing the siRNA of the invention is described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
- Suitable AAV vectors for expressing the siRNA of the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher KJ et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
- siRNA of the invention can be delivered to cultured cells infected with JCV, and the levels of target mRNA can be measured by Northern blot or dot blotting techniques, or by quantitative RT-PCR. Alternatively, the levels of agnoprotein or large T antigen in the cultured cells can be measured by ELISA or Western blot. Suitable protocols for infecting cultured cells with JCV, the delivery of siRNA to cultured cells, and assays for detecting protein and mRNA levels in cultured cells, are within the skill in the art (see, e.g., the Examples below).
- the siRNA of the invention target can cause the RNAi-mediated degradation of JCV agnoprotein gene or large T antigen gene mRNA, or alternative splice forms or mutants thereof.
- the siRNA of the invention comprises a target sequence in common with other primate polyomavirus genes (e.g., genes from BKV and/or SV40), the siRNA can cause the degradation of mRNA produced from these other primate polyomavirus genes as well. Degradation of the target mRNA by the present siRNA reduces the production of a functional gene product from these genes, and also prevents or reduces expression of other JCV genes (such as the VPl gene).
- the invention provides a method of inhibiting expression of agnoprotein gene, large T antigen gene, or other JCV genes (such as VPl) in a subject, comprising administering an effective amount of an siRNA of the invention to the subject, such that the target mRNA is degraded.
- the invention also provides a method of inhibiting JCV virus infection and/or replication in a subject by the RNAi- mediated degradation of the target mRNA by the present siRNA.
- the siRNA of the invention can also be used to treat diseases associated with JCV or other primate polyomavirus infection, such as are known in the art.
- JCV infection is associated with cancer of central nervous system (CNS) origin (including medullablastoma and glioblastoma) the demyelinating disease PML.
- CNS central nervous system
- the invention provides a method of treating diseases associated with JCV, BKV and/or SV40 infection, comprising administering to a subject an effective amount of one or more siRNA of the invention.
- a "disease associated with JCV infection” means any disease or disorder which is correlated with the presence of an active or latent JCV infection.
- Exemplary diseases associated with JCV infection are CNS cancers such as medullablastoma and glioblastoma, and demyelinating diseases such as PML.
- a "disease associated with BKV infection” means any disease or disorder which is correlated with the presence of an active or latent BKV infection, and includes polyomavirus-associated neuropathy.
- a "disease associated with SV40 infection” means any disease or disorder which is correlated with the presence of an active or latent SV40 infection, and includes SV40-induced cancers such as mesothelioma.
- to "treat" a disease associated with JCV infection means that symptoms associated with the disease are do not worsen, are reduced or are prevented. Evaluation of symptoms of diseases associated with JCV infection are within the skill in the art.
- two or more siRNA comprising different target sequences in the JCV agnoprotein gene or large T antigen gene mRNA can be administered to the subject.
- two or more siRNA, each comprising target sequences from JCV agnoprotein gene and large T antigen gene mRNA are be administered to a subject.
- a "subject” includes a human being or non-human animal.
- the subject is a human being.
- an "effective amount" of the siRNA is an amount sufficient to cause RNAi-mediated degradation of the target mRNA, or an amount sufficient to treat a disease associated with JCV, BKV and/or SV40 infection in a subject.
- RNAi-mediated degradation of the target mRNA can be detected by measuring levels of the target mRNA or protein in the cells of a subject, using standard techniques for isolating and quantifying mRNA or protein as described above.
- the siRNA of the invention can mediate RNA interference in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the siRNA of the invention induces the RISC to degrade the target mRNA in a catalytic manner.
- primate polyomavirus e.g., JCV, BKV and/or SV40
- an effective amount of the siRNA of the invention to be administered to a given subject, by taking into account factors such as the size and weight of the subject; the extent of the JCV infection or disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic.
- an effective amount of the siRNA of the invention comprises an intercellular concentration at or near the neovascularization site of from about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50 nM, more preferably from about 2.5 nM to about 10 nM.
- Particularly preferred effective amounts of the siRNA of the invention can comprise an intercellular concentration at or near the neovascularization site of about 1 nM, about 5 nM, or about 25 nM. It is contemplated that greater or lesser effective amounts of siRNA can be administered.
- the siRNA of the invention can administered to a subject in combination with a pharmaceutical agent which is different from the present siRNA.
- the siRNA of the invention can be administered in combination with therapeutic methods currently employed for treating cancer or preventing tumor metastasis (e.g., radiation therapy, chemotherapy, and surgery).
- the siRNA of the invention is preferably administered to a subject in combination with radiation therapy, or in combination with chemotherapeutic agents such as cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.
- the siRNA can be administered to the subject either as naked siRNA, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the siRNA.
- Suitable delivery reagents for administration in conjunction with the present siRNA include the Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.
- a preferred delivery reagent is a liposome.
- Liposomes can aid in the delivery of the siRNA to a particular tissue, such as retinal or tumor tissue, and can also increase the blood half-life of the siRNA.
- Liposomes suitable for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9: 467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which are herein incorporated by reference.
- liposomes encapsulating the present siRNA comprise a ligand molecule that can target the liposome to cells which are infected with primate polyomavirus; e.g., JCV, BKV and/or SV40.
- primate polyomavirus e.g., JCV, BKV and/or SV40.
- the liposomes encapsulating the present siRNA are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example by having opsonization-inhibition moieties bound to the surface of the structure.
- a liposome of the invention can comprise both opsonization-inhibition moieties and a ligand.
- Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane.
- an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids.
- opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in U.S. Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference.
- MMS macrophage-monocyte system
- RES reticuloendothelial system
- Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature. Thus, tissue characterized by such microvasculature defects, for example solid tumors, will efficiently accumulate these liposomes; see Gabizon, et al. (1988), P.N.A.S., USA, J8: 6949-53. In addition, the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in the liver and spleen. Thus, liposomes of the invention that are modified with opsonization-inhibition moieties are particularly suited to deliver the present siRNA to tumor cells.
- Opsonization inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a number average molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons.
- Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GMi. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable.
- PEG polyethylene glycol
- PPG polypropylene glycol
- the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide.
- the opsonization inhibiting polymers can also be namral polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.
- the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof.
- Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes.”
- the opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques.
- an N- hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane.
- a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH and a solvent mixture such as tetrahydrofuran and water in a 30: 12 ratio at 60 °C.
- Recombinant plasmids which express siRNA of the invention are discussed above. Such recombinant plasmids can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.
- a suitable delivery reagent including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.
- Recombinant viral vectors which express siRNA of the invention are also discussed above, and methods for delivering such vectors to cells of a subject which are infected with JCV are within the skill in the art.
- the siRNA of the invention can be administered to the subject by any means suitable for delivering the siRNA to cells infected with primate polyomavirus; e.g., JCV, BKV and/or SV40.
- the siRNA can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
- Suitable enteral administration routes include oral, rectal, or intranasal delivery.
- Suitable parenteral administration routes include intravascular administration (e.g. intravenous bolus injection, intravenous infusion, intra- arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue administration (e.g., peri-tumoral and intra- tumoral injection, intra-retinal injection or subretinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct (e.g., topical) application to the area at or near the site of neovascularization, for example by a catheter or other placement device (e.g., a corneal pellet or a suppository, eye-dropper, or an implant comprising a porous, non-porous, or gelatinous material); and inhalation.
- intravascular administration e.g. intravenous bolus injection, intravenous infusion
- Suitable placement devices include the ocular implants described in U.S. Pat. Nos. 5,902,598 and 6,375,972, and the biodegradable ocular implants described in U.S. Pat. No 6,331,313, the entire disclosures of which are herein incorporated by reference. Such ocular implants are available from Control Delivery Systems, Inc. (Watertown, MA) and Oculex Pharmaceuticals, Inc. (Sunnyvale, CA).
- injections or infusions of the siRNA are given at or near the site of primate polyomavirus infection.
- the siRNA of the invention can be administered in a single dose or in multiple doses. Where the administration of the siRNA of the invention is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions.
- the siRNA can be administered to the subject once, such as by a single injection or deposition at or near the site of primate polyomavirus infection.
- the siRNA can be administered to a subject multiple times daily or weekly.
- the siRNA can be administered to a subject once weekly for a period of from about three to about twenty-eight weeks, more preferably from about seven to about ten weeks.
- the siRNA is injected at or near the site of primate polyomavirus infection once a week for seven weeks. It is understood that periodic administrations of the siRNA of the invention for an indefinite length of time may be necessary for subjects suffering from a demyelinating disease such as PML.
- a dosage regimen comprises multiple administrations or the administration of two or more siRNA, each of which comprise a different target sequence
- the effective amount of siRNA administered to the subject can comprise the total amount of siRNA administered over the entire dosage regimen.
- the siRNA of the invention are preferably formulated as pharmaceutical compositions prior to administering to a subject, according to techniques known in the art.
- Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free.
- pharmaceutical formulations include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
- the present pharmaceutical formulations comprise an siRNA of the invention (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a physiologically acceptable carrier medium.
- Preferred physiologically acceptable carrier media are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
- compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives.
- Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
- Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA- bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
- Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
- solid compositions conventional nontoxic solid carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
- a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10-95%), preferably 25%-75%, of one or more siRNA of the invention.
- a pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20% by weight, preferably 1%-10% by weight, of one or more siRNA of the invention encapsulated in a liposome as described above, and propellant.
- a carrier can also be included as desired; e.g., lecithin for intranasal delivery.
- T-antigen siRNA To examine the ability of T-antigen siRNA to suppress viral gene expression during the course of infection, primary human fetal astrocytes were infected with the Mad-4 strain of JCV and at days 1, 5, and 10 post-infection, cells were transfected with T-antigen siRNA oligonucleotides. Results from analysis of viral proteins at 15 days post-infection showed drastic suppression of T-antigen, a marginal decrease in Agnoprotein, and a noticeable reduction in the level of the viral capsid protein, VPl, in the siRNA-treated cells in comparison with control cells. Neither viral infection nor siRNA treatment influenced the level of grb-2 production.
- siRNA designed for Agnoprotein from the polyomavirus, BKV which shares a two nucleotide mismatch with the analogous region of the JCV genome failed to suppress expression of JCV Agnoprotein in the transfected cells, verifying the high level of specificity of the designed siRNAs.
- JCV Agnoprotein siRNA, but not BKV Agnoprotein siRNA drastically suppressed the production of JCV Agnoprotein in JCV infected human fetal astrocytes.
- the extinction of Agnoprotein by siRNA affected production of the viral early protein, T- antigen. This observation suggests that Agnoprotein promotes the expression of T-antigen. Also, a noticeable decrease in the level of VP 1 was detected in cells treated with JCV Agnoprotein siRNA.
- Agnoprotein was decreased in the infected cells upon co-treatment of cells with both siRNAs.
- Treatment of the cells with T-antigen siRNA caused a drastic decrease in the expression of both T-antigen and VP I, but had a marginal effect on Agnoprotein production.
- Treatment of cells with Agnoprotein siRNA resulted in a major decline in the levels of Agnoprotein and VPl and resulted in suppression of T-antigen appearance in the nuclei of some, but not all, infected cells.
- the observed events were specific, as under similar conditions, BKV Agnoprotein siRNA had no effect on the production of T-antigen, VPl, or Agnoprotein.
- RNA interference which prevents the expression of genes by using small molecules, such as small interfering RNAs and siRNAs, has recently received special attention due to their ability to effect viral gene expression (Gitlin et al, 2002; Jacque et al, 2002; Ying et al, 2003; Chang and Taylor, 2003).
- siRNAs can be incorporated into a protein complex that recognizes and cleaves target mRNAs (Gitlin and Andino, 2003).
- T-antigen siRNA exhibited a more robust effect in infected cells, it should be noted that in the transient transfection assay, siRNA was transfected 24 h after the T-antigen plasmid and therefore may not have been delivered into the identical cells receiving the T-antigen expression vector. Furthermore, studies in stable cell lines show this siRNA completely abrogates T-antigen expression (data not shown).
- siRNA for targeting Agnoprotein production and showed that its use can also decrease the level of viral early and late protein production by more than 50%.
- siRNA we developed siRNA for targeting Agnoprotein production and showed that its use can also decrease the level of viral early and late protein production by more than 50%.
- VPl the major capsid protein
- VPl expression can be blocked via targeting of T-antigen and Agnoprotein, thus abrogating productive infection.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicinal Preparation (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2005244827A AU2005244827A1 (en) | 2004-05-12 | 2005-05-12 | Compositions and methods for siRNA inhibition of primate polyomavirus genes |
EP05749291A EP1753292A4 (en) | 2004-05-12 | 2005-05-12 | COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF PRIMATE POLYOMAVIRUS GENES |
CA002566462A CA2566462A1 (en) | 2004-05-12 | 2005-05-12 | Compositions and methods for sirna inhibition of primate polyomavirus genes |
US11/596,479 US20070249552A1 (en) | 2004-05-12 | 2005-05-12 | Compositions and Methods for Sirna Inhibition of Primate Polyomavirus Genes |
JP2007513352A JP2007536937A (en) | 2004-05-12 | 2005-05-12 | Compositions and methods for siRNA interference of primate polyomavirus genes |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US57034504P | 2004-05-12 | 2004-05-12 | |
US60/570,345 | 2004-05-12 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2005112636A2 true WO2005112636A2 (en) | 2005-12-01 |
WO2005112636A3 WO2005112636A3 (en) | 2007-03-15 |
Family
ID=35428772
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2005/016597 WO2005112636A2 (en) | 2004-05-12 | 2005-05-12 | COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF PRIMATE POLYOMAVIRUS GENES |
Country Status (6)
Country | Link |
---|---|
US (1) | US20070249552A1 (en) |
EP (1) | EP1753292A4 (en) |
JP (1) | JP2007536937A (en) |
AU (1) | AU2005244827A1 (en) |
CA (1) | CA2566462A1 (en) |
WO (1) | WO2005112636A2 (en) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007127919A2 (en) | 2006-04-28 | 2007-11-08 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of a gene from the jc virus |
US8138161B2 (en) | 2004-10-01 | 2012-03-20 | Novartis Vaccines And Diagnostics, Inc. | Modified small interfering RNA molecules and methods of use |
WO2012143427A1 (en) * | 2011-04-19 | 2012-10-26 | Santaris Pharma A/S | Anti polyomavirus compounds |
CN102985131A (en) * | 2010-04-28 | 2013-03-20 | 金伯利-克拉克环球有限公司 | Medical devices for delivery of siRNA |
US8765704B1 (en) | 2008-02-28 | 2014-07-01 | Novartis Ag | Modified small interfering RNA molecules and methods of use |
WO2015042466A3 (en) * | 2013-09-19 | 2015-08-27 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Compositions and methods for inhibiting jc virus (jcv) |
US20210010004A1 (en) * | 2018-03-02 | 2021-01-14 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Inhibition of polyomavirus replication |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100284921A1 (en) * | 2009-05-08 | 2010-11-11 | Temple University - Of The Commonwealth System Of Higher Education | Targeted nanoparticles for intracellular cancer therapy |
CN102971037B (en) | 2010-04-28 | 2016-05-25 | 金伯利-克拉克环球有限公司 | For sending the device of medicine for treating rheumatoid arthritis |
WO2012046149A1 (en) | 2010-04-28 | 2012-04-12 | Kimberly-Clark Worldwide, Inc. | Method for increasing permeability of an epithelial barrier |
CN102958556B (en) | 2010-04-28 | 2017-02-15 | 金伯利-克拉克环球有限公司 | Nanopatterned medical device with enhanced cellular interaction |
CN103906549A (en) | 2011-10-27 | 2014-07-02 | 金伯利-克拉克环球有限公司 | Implantable devices for delivery of bioactive agents |
RU2630610C2 (en) | 2011-10-27 | 2017-09-11 | Кимберли-Кларк Ворлдвайд, Инк. | Transdermal delivery of high-binded bioactive agents |
US20170246439A9 (en) | 2011-10-27 | 2017-08-31 | Kimberly-Clark Worldwide, Inc. | Increased Bioavailability of Transdermally Delivered Agents |
US20190038770A1 (en) * | 2016-01-25 | 2019-02-07 | Excision Biotherapeutics,Inc. | Rna guided eradication of human jc virus and other polyomaviruses |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CZ333294A3 (en) * | 1992-07-02 | 1995-07-12 | Hybridon | Self-stabilized oligonucleotide and inhibition method of virus gene or pathogenic organism or cellular gene expression |
WO2003070918A2 (en) * | 2002-02-20 | 2003-08-28 | Ribozyme Pharmaceuticals, Incorporated | Rna interference by modified short interfering nucleic acid |
JP4840792B2 (en) * | 2001-11-22 | 2011-12-21 | 独立行政法人科学技術振興機構 | Treatment of PML for JC virus agno |
-
2005
- 2005-05-12 JP JP2007513352A patent/JP2007536937A/en active Pending
- 2005-05-12 US US11/596,479 patent/US20070249552A1/en not_active Abandoned
- 2005-05-12 CA CA002566462A patent/CA2566462A1/en not_active Abandoned
- 2005-05-12 EP EP05749291A patent/EP1753292A4/en not_active Withdrawn
- 2005-05-12 WO PCT/US2005/016597 patent/WO2005112636A2/en active Application Filing
- 2005-05-12 AU AU2005244827A patent/AU2005244827A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
See references of EP1753292A4 * |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8138161B2 (en) | 2004-10-01 | 2012-03-20 | Novartis Vaccines And Diagnostics, Inc. | Modified small interfering RNA molecules and methods of use |
US9084808B2 (en) | 2004-10-01 | 2015-07-21 | Arrowhead Research Corporation | Modified small interfering RNA molecules and methods of use |
EP2013222A2 (en) * | 2006-04-28 | 2009-01-14 | Alnylam Pharmaceuticals Inc. | Compositions and methods for inhibiting expression of a gene from the jc virus |
EP2013222A4 (en) * | 2006-04-28 | 2009-10-21 | Alnylam Pharmaceuticals Inc | Compositions and methods for inhibiting expression of a gene from the jc virus |
US7691824B2 (en) * | 2006-04-28 | 2010-04-06 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of a gene from the JC virus |
WO2007127919A2 (en) | 2006-04-28 | 2007-11-08 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of a gene from the jc virus |
US8765704B1 (en) | 2008-02-28 | 2014-07-01 | Novartis Ag | Modified small interfering RNA molecules and methods of use |
CN102985131A (en) * | 2010-04-28 | 2013-03-20 | 金伯利-克拉克环球有限公司 | Medical devices for delivery of siRNA |
WO2012143427A1 (en) * | 2011-04-19 | 2012-10-26 | Santaris Pharma A/S | Anti polyomavirus compounds |
WO2015042466A3 (en) * | 2013-09-19 | 2015-08-27 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Compositions and methods for inhibiting jc virus (jcv) |
US10036020B2 (en) | 2013-09-19 | 2018-07-31 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Compositions and methods for inhibiting JC virus (JCV) |
US20210010004A1 (en) * | 2018-03-02 | 2021-01-14 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Inhibition of polyomavirus replication |
US11661602B2 (en) * | 2018-03-02 | 2023-05-30 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Inhibition of polyomavirus replication |
Also Published As
Publication number | Publication date |
---|---|
CA2566462A1 (en) | 2005-12-01 |
EP1753292A2 (en) | 2007-02-21 |
WO2005112636A3 (en) | 2007-03-15 |
AU2005244827A1 (en) | 2005-12-01 |
EP1753292A4 (en) | 2007-09-26 |
US20070249552A1 (en) | 2007-10-25 |
JP2007536937A (en) | 2007-12-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20070249552A1 (en) | Compositions and Methods for Sirna Inhibition of Primate Polyomavirus Genes | |
US8541384B2 (en) | Compositions and methods for siRNA inhibition of angiogenesis | |
US7473525B2 (en) | Compositions and methods for inhibiting expression of anti-apoptotic genes | |
US7872118B2 (en) | siRNA and methods of manufacture | |
US20100151007A1 (en) | Compositions and methods for selective inhibition of vegf | |
US8227444B2 (en) | Compositions and methods for inhibition of VEGF | |
WO2004094606A2 (en) | COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF ANGIOPOIETIN 1 AND 2 AND THEIR RECEPTOR TIE2 | |
US20210348167A1 (en) | siNA MOLECULES, METHODS OF PRODUCTION AND USES THEREOF | |
US20210332364A1 (en) | siNA MOLECULES, METHODS OF PRODUCTION AND USES THEREOF |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2007513352 Country of ref document: JP Ref document number: 2566462 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 11596479 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2005244827 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2005749291 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2005244827 Country of ref document: AU Date of ref document: 20050512 Kind code of ref document: A |
|
WWP | Wipo information: published in national office |
Ref document number: 2005244827 Country of ref document: AU |
|
WWP | Wipo information: published in national office |
Ref document number: 2005749291 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 11596479 Country of ref document: US |