CN101426913A - 使用siRNA敲减基因表达和改善实体器官和细胞移植的组合物和方法 - Google Patents
使用siRNA敲减基因表达和改善实体器官和细胞移植的组合物和方法 Download PDFInfo
- Publication number
- CN101426913A CN101426913A CNA2006800520917A CN200680052091A CN101426913A CN 101426913 A CN101426913 A CN 101426913A CN A2006800520917 A CNA2006800520917 A CN A2006800520917A CN 200680052091 A CN200680052091 A CN 200680052091A CN 101426913 A CN101426913 A CN 101426913A
- Authority
- CN
- China
- Prior art keywords
- target
- sequence
- polynucleotide
- seeking
- organ
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108020004459 Small interfering RNA Proteins 0.000 title claims abstract description 181
- 210000000056 organ Anatomy 0.000 title claims abstract description 90
- 238000000034 method Methods 0.000 title claims abstract description 76
- 239000000203 mixture Substances 0.000 title claims abstract description 45
- 238000002054 transplantation Methods 0.000 title claims abstract description 23
- 230000014509 gene expression Effects 0.000 title claims description 27
- 239000007787 solid Substances 0.000 title description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 53
- 108091033319 polynucleotide Proteins 0.000 claims description 72
- 102000040430 polynucleotide Human genes 0.000 claims description 72
- 239000002157 polynucleotide Substances 0.000 claims description 72
- 239000002773 nucleotide Substances 0.000 claims description 44
- 125000003729 nucleotide group Chemical group 0.000 claims description 44
- 210000003734 kidney Anatomy 0.000 claims description 43
- 210000004027 cell Anatomy 0.000 claims description 41
- 230000002519 immonomodulatory effect Effects 0.000 claims description 17
- 230000001900 immune effect Effects 0.000 claims description 16
- 239000002105 nanoparticle Substances 0.000 claims description 13
- 108010000134 Vascular Cell Adhesion Molecule-1 Proteins 0.000 claims description 12
- 102100023543 Vascular cell adhesion protein 1 Human genes 0.000 claims description 10
- 230000010412 perfusion Effects 0.000 claims description 9
- 102000004889 Interleukin-6 Human genes 0.000 claims description 8
- 108090001005 Interleukin-6 Proteins 0.000 claims description 8
- 210000000496 pancreas Anatomy 0.000 claims description 8
- 108010002352 Interleukin-1 Proteins 0.000 claims description 7
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 7
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 7
- 108090001007 Interleukin-8 Proteins 0.000 claims description 6
- 210000002216 heart Anatomy 0.000 claims description 6
- 210000004072 lung Anatomy 0.000 claims description 6
- 229920000642 polymer Polymers 0.000 claims description 6
- 238000003860 storage Methods 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 5
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 5
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 claims description 5
- 102100037850 Interferon gamma Human genes 0.000 claims description 5
- 108010074328 Interferon-gamma Proteins 0.000 claims description 5
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 5
- 210000004185 liver Anatomy 0.000 claims description 5
- 108010028780 Complement C3 Proteins 0.000 claims description 4
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 claims description 4
- 102000004890 Interleukin-8 Human genes 0.000 claims description 4
- 108700018351 Major Histocompatibility Complex Proteins 0.000 claims description 4
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 claims description 4
- 210000002919 epithelial cell Anatomy 0.000 claims description 4
- 229940121354 immunomodulator Drugs 0.000 claims description 4
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 claims description 4
- 108010021064 CTLA-4 Antigen Proteins 0.000 claims description 3
- 210000004087 cornea Anatomy 0.000 claims description 3
- 210000003038 endothelium Anatomy 0.000 claims description 3
- 239000002955 immunomodulating agent Substances 0.000 claims description 3
- 210000000265 leukocyte Anatomy 0.000 claims description 3
- 230000002829 reductive effect Effects 0.000 claims description 3
- 210000000813 small intestine Anatomy 0.000 claims description 3
- 229940045513 CTLA4 antagonist Drugs 0.000 claims description 2
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 claims description 2
- 210000004204 blood vessel Anatomy 0.000 claims description 2
- 229940125645 monoclonal antibody drug Drugs 0.000 claims description 2
- 210000004165 myocardium Anatomy 0.000 claims description 2
- 229940126586 small molecule drug Drugs 0.000 claims description 2
- 210000004509 vascular smooth muscle cell Anatomy 0.000 claims description 2
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical group S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 claims 2
- 102000015271 Intercellular Adhesion Molecule-1 Human genes 0.000 claims 2
- 229940100601 interleukin-6 Drugs 0.000 claims 2
- XKTZWUACRZHVAN-VADRZIEHSA-N interleukin-8 Chemical group C([C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](NC(C)=O)CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N1[C@H](CCC1)C(=O)N1[C@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC(O)=CC=1)C(=O)N[C@H](CO)C(=O)N1[C@H](CCC1)C(N)=O)C1=CC=CC=C1 XKTZWUACRZHVAN-VADRZIEHSA-N 0.000 claims 2
- 102000016918 Complement C3 Human genes 0.000 claims 1
- 238000007654 immersion Methods 0.000 claims 1
- 229960003130 interferon gamma Drugs 0.000 claims 1
- 229940096397 interleukin-8 Drugs 0.000 claims 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 16
- 108020004999 messenger RNA Proteins 0.000 description 16
- 108091034117 Oligonucleotide Proteins 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 239000000126 substance Substances 0.000 description 14
- 230000000694 effects Effects 0.000 description 13
- 206010063837 Reperfusion injury Diseases 0.000 description 12
- 208000028867 ischemia Diseases 0.000 description 12
- 108090000765 processed proteins & peptides Proteins 0.000 description 12
- 230000002068 genetic effect Effects 0.000 description 11
- 230000000295 complement effect Effects 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 238000001890 transfection Methods 0.000 description 9
- 241000700159 Rattus Species 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 102100022133 Complement C3 Human genes 0.000 description 6
- 102100037877 Intercellular adhesion molecule 1 Human genes 0.000 description 6
- 102000000589 Interleukin-1 Human genes 0.000 description 6
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000000692 anti-sense effect Effects 0.000 description 5
- 229920006317 cationic polymer Polymers 0.000 description 5
- 239000012141 concentrate Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000008728 vascular permeability Effects 0.000 description 5
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 4
- 101000901154 Homo sapiens Complement C3 Proteins 0.000 description 4
- 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 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
- 238000013459 approach Methods 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000011010 flushing procedure Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 230000028993 immune response Effects 0.000 description 4
- 239000003018 immunosuppressive agent Substances 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 208000037816 tissue injury Diseases 0.000 description 4
- 208000003918 Acute Kidney Tubular Necrosis Diseases 0.000 description 3
- 101150013553 CD40 gene Proteins 0.000 description 3
- 101000599852 Homo sapiens Intercellular adhesion molecule 1 Proteins 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 206010038540 Renal tubular necrosis Diseases 0.000 description 3
- 108091028664 Ribonucleotide Proteins 0.000 description 3
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 238000001994 activation Methods 0.000 description 3
- 230000000735 allogeneic effect Effects 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 239000005547 deoxyribonucleotide Substances 0.000 description 3
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 230000000302 ischemic effect Effects 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 230000008520 organization Effects 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 238000002823 phage display Methods 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000002336 ribonucleotide Substances 0.000 description 3
- 125000002652 ribonucleotide group Chemical group 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 101150071258 C3 gene Proteins 0.000 description 2
- 108010029697 CD40 Ligand Proteins 0.000 description 2
- 102100032937 CD40 ligand Human genes 0.000 description 2
- 102100031506 Complement C5 Human genes 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 108010074864 Factor XI Proteins 0.000 description 2
- 208000009329 Graft vs Host Disease Diseases 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- 241000702617 Human parvovirus B19 Species 0.000 description 2
- 101150050263 ICAM1 gene Proteins 0.000 description 2
- MBBZMMPHUWSWHV-BDVNFPICSA-N N-methylglucamine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO MBBZMMPHUWSWHV-BDVNFPICSA-N 0.000 description 2
- 208000003670 Pure Red-Cell Aplasia Diseases 0.000 description 2
- 102000039471 Small Nuclear RNA Human genes 0.000 description 2
- 108091027967 Small hairpin RNA Proteins 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 101150097457 Vcam1 gene Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000004913 activation Effects 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
- 238000011316 allogeneic transplantation Methods 0.000 description 2
- 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 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
- 150000001875 compounds Chemical class 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 208000024908 graft versus host disease Diseases 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 229920001477 hydrophilic polymer Polymers 0.000 description 2
- 230000002631 hypothermal effect Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 229960003444 immunosuppressant agent Drugs 0.000 description 2
- 230000001861 immunosuppressant effect Effects 0.000 description 2
- 229940026289 infasurf Drugs 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 229960003194 meglumine Drugs 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- -1 polyoxyethylene Polymers 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000002516 radical scavenger Substances 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 108091029842 small nuclear ribonucleic acid Proteins 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 2
- 229940045145 uridine Drugs 0.000 description 2
- 230000001196 vasorelaxation Effects 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- VEEGZPWAAPPXRB-BJMVGYQFSA-N (3e)-3-(1h-imidazol-5-ylmethylidene)-1h-indol-2-one Chemical compound O=C1NC2=CC=CC=C2\C1=C/C1=CN=CN1 VEEGZPWAAPPXRB-BJMVGYQFSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000707825 Argyrosomus regius Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 101800004538 Bradykinin Proteins 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 101800001654 C5a anaphylatoxin Proteins 0.000 description 1
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 1
- 102100030563 Coagulation factor XI Human genes 0.000 description 1
- 108010034753 Complement Membrane Attack Complex Proteins 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 108010080865 Factor XII Proteins 0.000 description 1
- 102000000429 Factor XII Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229940122498 Gene expression inhibitor Drugs 0.000 description 1
- QXZGBUJJYSLZLT-UHFFFAOYSA-N H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH Natural products NC(N)=NCCCC(N)C(=O)N1CCCC1C(=O)N1C(C(=O)NCC(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CO)C(=O)N2C(CCC2)C(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CCCN=C(N)N)C(O)=O)CCC1 QXZGBUJJYSLZLT-UHFFFAOYSA-N 0.000 description 1
- 108010042653 IgA receptor Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 102100026720 Interferon beta Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 108010025252 Kassinin Proteins 0.000 description 1
- 102100035792 Kininogen-1 Human genes 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108010087870 Mannose-Binding Lectin Proteins 0.000 description 1
- 102100026553 Mannose-binding protein C Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000006404 Mitochondrial Proteins Human genes 0.000 description 1
- 108010058682 Mitochondrial Proteins Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010053159 Organ failure Diseases 0.000 description 1
- 208000031816 Pathologic Dilatation Diseases 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 229930182556 Polyacetal Natural products 0.000 description 1
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 241001558929 Sclerotium <basidiomycota> Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 206010060872 Transplant failure Diseases 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 102000018594 Tumour necrosis factor Human genes 0.000 description 1
- 108050007852 Tumour necrosis factor Proteins 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 229940121369 angiogenesis inhibitor Drugs 0.000 description 1
- 239000004037 angiogenesis inhibitor Substances 0.000 description 1
- 230000003409 anti-rejection Effects 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- QXZGBUJJYSLZLT-FDISYFBBSA-N bradykinin Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CO)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CCC1 QXZGBUJJYSLZLT-FDISYFBBSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 229940026290 calfactant Drugs 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 229920003118 cationic copolymer Polymers 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000004671 cell-free system Anatomy 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002975 chemoattractant Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 102000006834 complement receptors Human genes 0.000 description 1
- 108010047295 complement receptors Proteins 0.000 description 1
- 230000004154 complement system Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 1
- 229960002986 dinoprostone Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 210000000887 face Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000010231 histologic analysis Methods 0.000 description 1
- 102000057770 human C3 Human genes 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 239000012642 immune effector Substances 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002584 immunomodulator Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 229940124589 immunosuppressive drug Drugs 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- TYYBFXNZMFNZJT-UHFFFAOYSA-N ioxaglic acid Chemical compound CNC(=O)C1=C(I)C(N(C)C(C)=O)=C(I)C(C(=O)NCC(=O)NC=2C(=C(C(=O)NCCO)C(I)=C(C(O)=O)C=2I)I)=C1I TYYBFXNZMFNZJT-UHFFFAOYSA-N 0.000 description 1
- 229960001707 ioxaglic acid Drugs 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- GDBREXONAMPGBA-FJCMUPJRSA-N kassinin Chemical compound C([C@@H](C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)C(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=CC=C1 GDBREXONAMPGBA-FJCMUPJRSA-N 0.000 description 1
- 230000013580 kinin cascade Effects 0.000 description 1
- 150000002617 leukotrienes Chemical class 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 229940066294 lung surfactant Drugs 0.000 description 1
- 239000003580 lung surfactant Substances 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000004768 organ dysfunction Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000006320 pegylation Effects 0.000 description 1
- 229920006324 polyoxymethylene Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 238000003375 selectivity assay Methods 0.000 description 1
- 229940076279 serotonin Drugs 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000016160 smooth muscle contraction Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- MBVDCEVFLAONHO-UHFFFAOYSA-M sodium;3-[[2-[[3-[acetyl(methyl)amino]-2,4,6-triiodo-5-(methylcarbamoyl)benzoyl]amino]acetyl]amino]-5-(2-hydroxyethylcarbamoyl)-2,4,6-triiodobenzoate Chemical compound [Na+].CNC(=O)C1=C(I)C(N(C)C(C)=O)=C(I)C(C(=O)NCC(=O)NC=2C(=C(C(=O)NCCO)C(I)=C(C([O-])=O)C=2I)I)=C1I MBVDCEVFLAONHO-UHFFFAOYSA-M 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1136—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 growth factors, growth regulators, cytokines, lymphokines or hormones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—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
-
- 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
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/31—Combination therapy
-
- 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
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/32—Special delivery means, e.g. tissue-specific
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Endocrinology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Transplantation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Materials For Medical Uses (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明描述了组合物和方法,它们使用siRNA靶向在所移植器官或组织的细胞中表达的各种基因和/或在宿主中表达的基因,以提高移植成功率。
Description
本申请要求美国临时申请号60/741,157的优先权,其全部公开内容引入这里作为参考。
发明领域
本发明提供了使用siRNA介导的基因表达下调以预防实体器官或组织移植中的同种异体移植排斥或异种移植排斥和缺血/再灌注损伤的组合物和方法。
发明背景
实体器官移植是治疗末期器官衰竭的唯一有效疗法(1,2)。世界各地移植程序已经变得日益成功且这种操作变得愈加常规(3,4)。尽管一年生存率的结果给人深刻印象,但是器官移植仍然面临严重的问题。免疫系统成为所移植器官长期存活的最显著障碍。如果不用有效的免疫抑制剂终生治疗以阻止免疫反应的话,器官移植物将始终被排斥。然而,当前的抗排斥药物非选择性地降低全身免疫力和长期增加机会感染和肿瘤发生的风险。因此,正在寻求替换的策略。
过去十年中分子技术的进步使我们对引起免疫反应和缺血/再灌注损伤所需的信号都加深了了解。设计靶向这些新信号的试剂提供了它们会最终允许移植器官长期存在且不接受药物的希望。
移植免疫学是指同种异体移植物或异种移植物从供体取出,然后移植给受体后所发生的多方面的事件过程。在移植物和移植部位,组织都受损。之后立即发生炎症反应,如同生物化学级联的激活。当抗原被识别时一系列特异性和非特异性的细胞反应随之发生。最后,损害通过组织修复和强化被控制;如果损害是非病理性的,则移植物存活。
与抗原无关的组织损伤的原因(即缺血、低体温、再灌注损伤)是当收获移植物时血液供应的机械性创伤以及破坏的结果。
相反,抗原依赖性的组织损伤的原因包括免疫介导的损伤。巨噬细胞释放细胞因子(例如,肿瘤坏死因子、白介素-1),这通过刺激炎性内皮反应使炎症强度加强;这些内皮改变有助于募集大量T细胞到达移植部位。损伤组织释放触发几个生物化学级联的促炎介质(例如,Hageman因子[因子XII])。凝血级联诱发血纤维蛋白和几种相关的血纤维蛋白肽,它们促进局部血管通透性并吸引嗜中性白细胞和巨噬细胞。激肽级联主要产生缓激肽,其促进血管舒张、平滑肌收缩和增加血管通透性。
抗体-抗原复合物(即免疫复合物)的形成激活补体系统的传统途径。当C1q对接到免疫复合物内的抗体上时,它通过传统途径激发激活过程,同时补体因子C3可识别受损细胞表面作为旁路途径激活的受体。激活的补体通过膜攻击复合物(例如,C5b、C6、C7、C8、C9)和细胞结合配体例如C4b和C3b(其激活白细胞承载补体受体)的沉积引起损害。此外,生物活性过敏毒素C5a和C3a的产生引起炎性细胞流入和激活。这些趋化物也启动肥大细胞脱粒,其释放几种介质。组胺和5-羟色胺增加血管通透性。前列腺素E2促进血管舒张和血管通透性。白细胞三烯B4和D2促进白细胞蓄积和血管通透性。激活补体的另一种方式是通过组织缺血和再灌注,其使磷脂和线粒体蛋白暴露。这些副产物通过结合C1q或甘露糖结合凝集素或因子C3b而直接激活补体。
目前,成功的同种异体移植需要全身使用免疫抑制药物。由于毒性和对癌症和感染的易感性增加,这些可引起严重的发病。局部产生的免疫抑制分子局限于移植部位会降低对常规、通用的免疫抑制治疗的需要,并由此引起较少的副作用。这在疾病,如1型糖尿病中特别突出,这种疾病不立即危及生命,然而胰岛同种异体移植可以达到治愈。当联合使用抗体时,抗CD4策略可能越发有效;相似的策略也可以预防异种移植排斥。抑制宿主的免疫反应也会增加癌症的风险。抑制免疫反应以避免移植排斥和移植物抗宿主疾病(GVHD)的尝试使身体与传染性物质(例如细菌、病毒、真菌等等)斗争的能力削弱。
RNA干扰(RNAi)化合物,中间体短干扰RNA寡核苷酸(siRNA)提供了独特的策略,在同一治疗中联合使用多个siRNA双链体以靶向多个引起疾病的基因,因为所有siRNA双链体在化学上是同质的,具有相同来源和相同制造方法(5,6,7,8)。预计这种siRNA抑制剂具有好得多的临床功效,毒性和安全忧虑最小。遗传修饰对于器官移植是有前景的治疗策略。基于吸引人的沉默特定基因表达的RNA干扰技术(9,10),siRNA疗法可以代表预防缺血/再灌注损伤以及移植受体中的器官排斥的吸引人和有效的方法。
发明概述
本发明提供了寻靶多核苷酸,其靶向待捐献给受体的器官细胞中存在的免疫调节或免疫效应基因。这些多核苷酸的靶可以来源于表1-15(参见下文)中列出的免疫调节和免疫效应基因的序列。例如,寻靶多核苷酸可以靶向C3、ICAM1、VCAM-1、IFN-γ、IL-1、IL-6、IL-8、TNF-α、CD80、CD86、MHC-II、MHC-I、CD28、CTLA-4或PV-B19基因中的序列。寻靶多核苷酸可以包含靶向表1-15中所列一个或多个序列的siRNA双链体。寻靶多核苷酸可以是单链线性多核苷酸、双链线性多核苷酸或发夹多核苷酸。
本发明还提供了通过在将器官移植入受体前,使器官与包含本发明的寻靶多核苷酸的组合物接触来抑制移植器官排斥的方法。该方法可有效下调或抑制在移植前贮藏期间器官或器官的细胞中免疫调节或免疫效应靶基因的表达。在一个实施方案中,用包含本发明的寻靶多核苷酸的组合物灌注该器官。在另一个实施方案中,将该器官浸泡或浸没在包含本发明的寻靶多核苷酸的组合物中。还可以将该组合物给予器官受体。在本发明的一些实施方案中,该器官可以是受体自己的器官。所述器官的受体可以是人。与包含本发明的寻靶多核苷酸的组合物接触的器官、组织和细胞包括肾、肝、肺、胰、心脏、小肠、角膜、上皮细胞、血管内皮、血管平滑肌细胞、心肌和移植时器官中居留的过客白细胞。
包含本发明的寻靶多核苷酸的组合物还可以包含载体,包括但不限于灌注流体、高渗枸橼酸盐溶液、PolyTran聚合物溶液、TargeTran纳米颗粒溶液或威斯康星大学溶液(University of Wisconsinsolution)。该组合物还可以包含小分子药物、单克隆抗体药物,及其他免疫调节剂。在一些实施方案中,该组合物包含多个本发明的寻靶多核苷酸。组合物可以含有许多可靶向多个基因序列的本发明的寻靶多核苷酸。在一个实施方案中,该寻靶多核苷酸是靶向C3、TNF-α和IL-8基因序列的混合物。
附图简述
图1是显示大鼠肾细胞中C3 mRNA相对表达的柱状图。用IL-1和IL-6刺激细胞以增加C3表达。三个候选C3 siRNA序列(C3-1、C3-2、C3-3)或FITC标记的混杂siRNA以各种浓度转染到细胞中。用Lipofectamine和无siRNA(+lipofectamine)处理一组细胞,而刺激另一组细胞产生C3,既不用Lipofectamine也不用siRNA(-lipofectamine)处理。转染后48小时,通过实时PCR测定细胞中C3 mRNA水平。虚线表示未受刺激的细胞的C3表达。该实验表明通过siRNA敲减基因的可行性和功效。C3-3 siRNA被选为候选者用于进一步实验。
图2是显示用IL-1和IL-6刺激以增加C3表达的大鼠肾细胞中C3 mRNA相对表达的柱状图。这些细胞也用各种浓度的C3-3候选序列进行转染。实时PCR测定刺激48小时后的C3 mRNA表达表明这个siRNA序列导致C3表达与未用siRNA处理的受刺激细胞相比降低。使测定标准化为细胞中未受刺激的C3 mRNA表达(虚线)。
图3是显示移植的大鼠肾脏中C3 mRNA的相对表达水平的柱状图。该肾脏在移植前,不用或用含有各种量的混杂或C3特异性siRNA的纳米颗粒来处理。每个数据点含有来自4个独立的肾脏的数据,且每个PCR反应一式三份进行。将这些实验条件下的C3 mRNA水平与正常非移植肾(NKC,正常肾对照)和未用siRNA处理的移植肾(ISCH,缺血对照)的C3 mRNA水平相比较。该图证明移植前用C3特异性siRNA处理的肾脏中的C3 mRNA水平与正常非移植肾和未用C3特异性siRNA处理的移植肾中的C3 mRNA水平相比较低。用各种比例的PolyTran包裹C3特异性siRNA,在图1中标记如下:C3:10μg C3 siRNA在PolyTran中以1:4.5;裸C3:10μg C3 siRNA无PolyTran;C3 3:1:10μg C3 siRNA在PolyTran中以1:3;C3 1.5:1:10μg C3 siRNA在PolyTran中以1:1.5。为了检验siRNA特异性的要求,在移植前,用混杂siRNA处理了两组肾:FITC:10μg混杂FITC标记的siRNA;SCRAM CON:10μg混杂未标记的siRNA。
图4是一组显示移植的大鼠肾的组织学分析的两个图。上图显示了移植后48小时未处理的肾。组织病理学揭示表示急性肾小管坏死(ATN)的普遍的肾小管衰减和肾小管扩张。这个特殊的病理与移植后移植组织最初无功能相关。下图描绘了移植后48小时,用C3 siRNA(与PolyTran以1:4.5的比例)预处理的肾。这个肾的组织病理学显示较少的ATN。
图5显示了呈现实验结果的两个柱状图,该实验用来鉴定可用于将含siRNA的纳米颗粒靶向特定器官的短肽。使用噬菌体展示来鉴定集中于移植肾的候选肽。图5的上图显示了一个实验的例证性数据,三轮噬菌体文库注射、回收和扩充后,从肾回收的噬菌体浓度(以每克组织的噬菌斑形成单位计)不断增加。在对照实验中,用链霉抗生物素蛋白作为噬菌体结合的靶(R3vsStrep)。图5的下图显示了第三轮生物淘选后,受体的移植肾(Tx肾)、正常肾(N肾)、胰、心脏和肺中回收的噬菌体数量。该数据显示了与从其他器官回收到的噬菌体数量相比的噬菌体驻入移植肾的选择性。
发明详述
如本文使用的“寡核苷酸”和基于此的类似术语是指由天然存在的核苷酸组成的短聚合物,以及由合成或修饰的核苷酸组成的聚合物,如前段刚刚描述过。寡核苷酸长度可以是10个或更多个核苷酸,或长度是15、或16、或17、或18、或19、或20个或更多个核苷酸,或长度是21、或22、或23、或24个或更多个核苷酸,或长度是25、或26、或27、或28、或29、或30个或更多个核苷酸,或长度是35个或更多个、40个或更多个、45个或更多个,直至大约50个核苷酸。siRNA寡核苷酸可以具有15至30个核苷酸之间任意数量的核苷酸。在许多实施方案中,siRNA可以具有21至25个核苷酸之间任意数量的核苷酸。
在许多实施方案中,siRNA可以具有两个平末端、或两个粘末端、或一个平末端和一个粘末端,或一个末端具有突出端。突出核苷酸的范围可以是一至四个或更多。
RNA干扰(RNAi)
根据本发明,免疫调节或免疫效应基因靶的基因表达被RNA干扰减弱。免疫调节或免疫效应基因的表达产物通过特异性双链siRNA核苷酸序列被靶向,该核苷酸序列与免疫调节或免疫效应基因靶序列的至少一个片段互补,该片段含有15至30个之间任意数量的核苷酸,或在多个情况下,其含有21至25个核苷酸之间任意数量的核苷酸,或更多个。这个靶可以存在于5′非翻译(UT)区、编码序列中、或3′UT区。参见例如PCT申请WO00/44895、WO99/32619、WO01/75164、WO01/92513、WO01/29058、WO01/89304、WO02/16620和WO02/29858,每篇文献的全部内容引入这里作为参考。
根据本发明的方法,使用siRNA抑制了免疫调节或免疫效应基因表达,和因此抑制由于不利的免疫反应造成的缺血/再灌注损伤或器官移植排斥。根据本发明的寻靶多核苷酸包括siRNA寡核苷酸。这种siRNA还可以通过化学合成与预定序列相同或相似的核苷酸序列来制备。参见例如Tuschl,Zamore,Lehmann,Bartel和Sharp(1999),Genes & Dev.13:3191-3197,其全部内容引入这里作为参考。可供选择地,寻靶siRNA可以使用靶向多核苷酸序列而获得,例如通过在无细胞体系,例如但不限于果蝇提取物中消化免疫调节或免疫效应核糖多核苷酸序列,或通过重组双链cRNA转录。
对于由相同长度的16-30nt正义链和16-30nt反义链组成的siRNA双链体,通常观察到有效的沉默。在多个实施方案中,siRNA成对双链体的每条链在3′末端具有另外2nt突出端。2nt 3′突出端的序列为siRNA靶识别特异性做出另外的微薄贡献。在一个实施方案中,3′突出端的核苷酸是核糖核苷酸。在可供选择的实施方案中,3′突出端的核苷酸是脱氧核糖核苷酸。3′脱氧核苷酸的使用使胞内稳定性增加。
本发明的重组表达载体当导入细胞内时,被加工以提供包含靶向器官内免疫调节或免疫效应基因的siRNA序列的RNA。这种载体可以是克隆入表达载体的DNA分子,包含以允许表达的方式侧接免疫调节或免疫效应基因寻靶序列的可操作连接的调节序列。在这个载体中,与靶RNA反义的RNA分子由第一启动子(例如克隆DNA3′的启动子序列)转录,和对于RNA靶是正义链的RNA分子由第二启动子(例如克隆DNA5′启动子序列)转录。然后正义和反义链在体内杂交产生靶向免疫调节或免疫效应基因序列的siRNA构建体。可供选择地,可以利用两个构建体产生siRNA构建体的正义和反义链。此外,克隆的DNA可以编码具有二级结构的转录物,其中单个转录物兼备来自靶基因或基因的正义和互补反义序列。在这个实施方案的一个实施例中,发夹RNAi产物与全部或部分靶基因相似。在另一实施例中,发夹RNAi产物是siRNA。侧接免疫调节或免疫效应基因序列的调节序列可以相同或可以不同,以致可以单独,或以时间或空间方式调节它们的表达。
在某些实施方案中,siRNA通过将免疫调节或免疫效应基因序列克隆入载体而被胞内转录,该载体含有例如来自较小核RNA(snRNA)U6或人RNA酶PRNAH1的RNA pol III转录单位。载体系统的一个实例是GeneSuppressorTM RNA干扰试剂盒(Imgenex Corp.)。U6和H1启动子是III型Pol III启动子的成员。U6样启动子的+1核苷酸总是鸟嘌呤核苷,而对于H1启动子的+1是腺嘌呤核苷。这些启动子的终止信号定义为五个连续的胸腺嘧啶核苷。转录物典型地在第二个尿嘧啶核苷后切开。在这个位置切开导致所表达siRNA中产生3′UU突出端,其类似于合成的siRNA的3′突出端。长度小于400个核苷酸的任何序列可以被这些启动子转录,因此它们是理想的适于表达例如大约50个核苷酸的RNA茎环转录物中大约21个核苷酸的siRNA。已经研究了RNAi的特性和影响siRNA功效的因素(参见例如Elbashir,Lendeckel和Tuschl(2001).Genes & Dev.15:188-200)。
寻靶多核苷酸长度通常是300个核苷酸或更少,并包括第一核苷酸序列,该序列靶向存在于捐献器官的细胞中或捐献器官一旦从供体取出而伴随的过客细胞中的基因序列,和当捐献器官引入受体受试者体内时,第一核苷酸序列引发免疫调节或免疫效应反应。在多核苷酸中,任何T(胸腺嘧啶核苷)或任何U(尿嘧啶核苷)可以任选被另一个取代。另外,在多核苷酸中,第一核苷酸序列由a)长度是15至30个的任意数量或更多个的核苷酸的序列,或b)a)给定序列的互补物组成。这种多核苷酸本文术语可以称作线性多核苷酸。单链多核苷酸常常是双链siRNA中的一条链。
在相关方面,如上所述的多核苷酸进一步包括通过环序列与第一核苷酸序列隔开的第二核苷酸序列,以致第二核苷酸序列
a)具有基本上与第一核苷酸序列相同的长度,和
b)基本上与第一核苷酸序列互补。
在这个后一结构中,术语称为发夹多核苷酸,第一核苷酸序列与第二核苷酸序列杂交形成发夹,该发夹的互补序列通过环序列连接。发夹多核苷酸被胞内消化形成双链siRNA。
在多个实施方案中,线性多核苷酸和发夹多核苷酸的靶是存在于捐献器官的细胞中,或捐献器官伴随的过客细胞中的基因序列,和第一核苷酸序列是
a)靶向选自所附表1-15给出序列的序列的寻靶序列;
b)比a)中给出序列更长的寻靶序列,其中寻靶序列靶向选自表1-15的序列;
c)a)或b)中给出序列的片段,其中该片段由长度至少15个核苷酸的相邻碱基序列组成,且最多比所选序列短一个碱基;
d)寻靶序列,其中直至5个核苷酸不同于a)-c)中给出的序列,或
e)a)至d)中给出的任何序列的互补物。
在线性多核苷酸或发夹多核苷酸的各种实施方案中,第一核苷酸序列的长度是21至25个任意数量的核苷酸。
在多个实施方案中,线性多核苷酸或发夹多核苷酸由寻靶序列组成,该寻靶序列靶向选自表1-15的序列,并任选包括与所选序列3′结合的二核苷酸突出端。在线性多核苷酸或发夹多核苷酸的又一另外的实施方案中,第一核苷酸序列3′末端的二核苷酸序列是TT、TU、UT或UU,和包括核糖核苷酸或脱氧核糖核苷酸或二者。在各种更多实施方案中,线性或发夹多核苷酸可以是DNA,或可以是RNA,或可以由脱氧核糖核苷酸和核糖核苷酸组成。
对特别是人基因特异性的siRNA寡核苷酸的示例序列列于下面表1a至15b。对于所列基因,该表包括21聚体,具有突出端,和25聚体,具有平末端。对移植供体是其他哺乳动物的基因特异性的潜在siRNA寡核苷酸序列应该参照相应的人基因来设计,但是应着眼于那些动物的基因序列。
表1:
C3基因中siRNA靶向的序列
C3基因:人补体成分3(C3),登录号:NM_000064,基因ID:4557384,选择25个siRNA候选物靶向下列基因序列:
表1a.23聚体序列:
# | 位置 | 序列 | GC% | 热力学值 |
1 | 1858-1880 | AAGGGCGTGTTCGTGCTGAATAA | 58 | -6.9(-13.5,-6.6) |
2 | 2797-2819 | AAGGCTGCCGTCTACCATCATTT | 58 | -5.3(-12.1,-6.8) |
3 | 3053-3075 | AACGGCTGAAGCACCTCATTGTG | 58 | -4.9(-11.7,-6.8) |
4 | 586-608 | AAGCAGGACTCCTTGTCTTCTCA | 53 | -4.6(-12.1,-7.5) |
5 | 4163-4185 | AACCAGCACCGGAAACAGAAAAG | 53 | -4.6(-11.5,-6.9) |
6 | 851-873 | AAGTGGAGGGAACTGCCTTTGTC | 58 | -4.5(-11.2,-6.7) |
7 | 805-827 | AAGGGCCTGGAGGTCACCATCAC | 68 | -4.4(-14.4,-10.0) |
8 | 4903-4925 | AAGCCCAACCTCAGCTACATCAT | 58 | -4.2(-13.2,-9.0) |
9 | 3572-3594 | AAGCAGGAGACTTCCTTGAAGCC | 53 | -4.0(-12.1,-8.1) |
10 | 1161-1183 | AATGCCCTTTGACCTCATGGTGT | 53 | -3.9(-12.7,-8.8) |
11 | 4118-4140 | AAGATCAACTCACCTGTAATAAA | 37 | -3.8(-9.1, -5.3) |
12 | 4663-4685 | AAGGCCTGTGAGCCAGGAGTGGA | 68 | -3.8(-13.2,-9.4) |
13 | 2598-2620 | AATCCGAGCCGTTCTCTACAATT | 53 | -3.7(-10.9,-7.2) |
14 | 925-947 | AAGCGCATTCCGATTGAGGATGG | 53 | -3.6(-12.5,-8.9) |
15 | 2848-2870 | AAGGTCGTGCCGGAAGGAATCAG | 63 | -3.5(-11.4,-7.9) |
16 | 2770-2792 | AAGACCGGCCTGCAGGAAGTGGA | 68 | -3.4(-11.4,-8.0) |
17 | 4843-4865 | AAGCTGGAGGAGAAGAAACACTA | 53 | -3.4(-12.1,-8.7) |
18 | 2097-2119 | AATGGACAAAGTCGGCAAGTACC | 47 | -3.4(-10.6,-7.2) |
19 | 4549-4571 | AAGGAGGATGGAAAGCTGAACAA | 53 | -3.3(-12.1,-8.8) |
20 | 4183-4205 | AAGAGGCCTCAGGATGCCAAGAA | 63 | -3.3(-12.3,-9.0) |
21 | 337-359 | AACAGGGAGTTCAAGTCAGAAAA | 47 | -3.2(-11.3,-8.1) |
22 | 1135-1157 | AAGACACCCAAGTACTTCAAACC | 42 | -3.2(-10.1,-6.9) |
23 | 673-695 | AAGATCCGAGCCTACTATGAAAA | 47 | -3.2(-10.3,-7.1) |
24 | 3890-3912 | AAGCCTTGGCTCAATACCAAAAG | 47 | -3.1(-10.9,-7.8) |
25 | 4570-4592 | AAGCTCTGCCGTGATGAACTGTG | 58 | -3.1(-11.1,-8.0) |
表1b.25聚体siRNA正义链序列:
1:2730 | CAAGUCCUCGUUGUCCGUUCCAUAU |
2:2798 | AGGCUGCCGUCUACCAUCAUUUCAU |
3:3504 | CAUCUCGCUGCAGGAGGCUAAAGAU |
4:4113 | GGCCAAAGAUCAACUCACCUGUAAU |
5:4199 | CCAAGAACACUAUGAUCCUUGAGAU |
6:4272 | CAUAUCCAUGAUGACUGGCUUUGCU |
7:4324 | GCCAAUGGUGUUGACAGAUACAUCU |
8:4357 | GAGCUGGACAAAGCCUUCUCCGAUA |
9:4672 | GAGCCAGGAGUGGACUAUGUGUACA |
10:5012 | CCUUCACCGAGAGCAUGGUUGUCUU |
表2:
ICAM1基因中siRNA靶向的序列:
ICAM1基因:人细胞间粘附分子1(CD54)、人鼻病毒受体(ICAM1),登录号:NM_000201,基因ID:4557877,选择19个siRNA靶向下列基因序列:
表2a.23聚体DNA正义链序列:
# | 位置 | 序列 | GC% | 热力学值 |
1 | 1567-1589 | AACCGCCAGCGGAAGATCAAGAA | 63 | -4.8(-12.9,-8.1) |
2 | 280-302 | AACCGGAAGGTGTATGAACTGAG | 53 | -3.8(-11.8,-8.0) |
3 | 641-663 | AAGGGCTGGAGCTGTTTGAGAAC | 58 | -3.7(-13.2,-9.5) |
4 | 1291-1313 | AATTCCCAGCAGACTCCAATGTG | 53 | -3.6(-10.4,-6.8) |
5 | 1533-1555 | AATGGGCACTGCAGGCCTCAGCA | 68 | -3.5(-12.7,-9.2) |
6 | 286-308 | AAGGTGTATGAACTGAGCAATGT | 42 | -3.4(-11.1,-7.7) |
7 | 1028-1050 | AAGGGACCGAGGTGACAGTGAAG | 63 | -2.9(-12.3,-9.4) |
8 | 311-333 | AAGAAGATAGCCAACCAATGTGC | 42 | -2.4(-8.9,-6.5) |
9 | 1210-1232 | AACCAGACCCGGGAGCTTCGTGT | 68 | -2.4(-10.4,-8.0) |
10 | 1327-1349 | AACCCATTGCCCGAGCTCAAGTG | 63 | -2.2(-10.3,-8.1) |
11 | 340-362 | AACTGCCCTGATGGGCAGTCAAC | 63 | -2.1(-11.5,-9.4) |
12 | 1012-1034 | AAGCCAGAGGTCTCAGAAGGGAC | 63 | -2.0(-12.1,-10.1) |
13 | 277-299 | AACAACCGGAAGGTGTATGAACT | 47 | -2.0(-9.1,-7.1) |
14 | 874-896 | AAGGCCTCAGTCAGTGTGACCGC | 63 | -2.0(-13.2,-11.2) |
15 | 323-345 | AACCAATGTGCTATTCAAACTGC | 37 | -1.7(-8.0,-6.3) |
16 | 133-155 | AATGCCCAGACATCTGTGTCCCC | 58 | -1.5(-12.7,-11.2) |
17 | 1048-1070 | AAGTGTGAGGCCCACCCTAGAGC | 63 | -1.5(-9.9,-8.4) |
18 | 943-965 | AACCAGAGCCAGGAGACACTGCA | 63 | -1.3(-10.4,-9.1) |
19 | 296-318 | AACTGAGCAATGTGCAAGAAGAT | 47 | -1.2(-9.2,-8.0) |
表2b.25聚体siRNA正义链序列:
1:300 | GAGCAAUGUGCAAGAAGAUAGCCAA |
2:316 | GAUAGCCAACCAAUGUGCUAUUCAA |
3:345 | CCCUGAUGGGCAGUCAACAGCUAAA |
4:1510 | ACUGUGGUAGCAGCCGCAGUCAUAA |
5:1544 | CAGGCCUCAGCACGUACCUCUAUAA |
6:1712 | CCACACUGAACAGAGUGGAAGACAU |
7:1783 | GCAUUGUCCUCAGUCAGAUACAACA |
8:1853 | CAUCUGAUCUGUAGUCACAUGACUA |
9:1884 | GAGGAAGGAGCAAGACUCAAGACAU |
10:1977 | GGACAUACAACUGGGAAAUACUGAA |
表3:
VCAM1基因中siRNA靶向的序列:
VCAM1基因:人血管细胞粘附分子1(VCAM1),转录变体2,mRNA。
登录号NM_080682.GI:18201908;转录变体1,mRNA。
登录号NM_001078,GI:18201907;人血管细胞粘附分子1mRNA,完整cds
gi|1179885|gb|M30257.1|HUMCAM1V[179885],人血管细胞粘附分子1mRNA,完整cds,
gi|340193|gb|M60335.1|HUMVCAM1[340193],人血管细胞粘附分子-1(VCAM1)基因,完整cds,
gi|340195|gb|M73255.1|
HUMVCAM1A[340195],人血管细胞粘附分子1(VCAM-1)的人mRNA,
gi|37648|emb|X53051.1|HSVCAM1[37648]
选择25个siRNA候选物靶向下列基因序列:
表3a.23聚体DNA正义链序列:
# | 位置 | 序列 | GC% | 热力学值 |
1 | 1858-1880 | AAGGGCGTGTTCGTGCTGAATAA | 58 | -6.9(-13.5,-6.6) |
2 | 2797-2819 | AAGGCTGCCGTCTACCATCATTT | 58 | -5.3(-12.1,-6.8) |
3 | 3053-3075 | AACGGCTGAAGCACCTCATTGTG | 58 | -4.9(-11.7,-6.8) |
4 | 586-608 | AAGCAGGACTCCTTGTCTTCTCA | 53 | -4.6(-12.1,-7.5) |
5 | 4163-4185 | AACCAGCACCGGAAACAGAAAAG | 53 | -4.6(-11.5,-6.9) |
6 | 851-873 | AAGTGGAGGGAACTGCCTTTGTC | 58 | -4.5(-11.2,-6.7) |
7 | 805-827 | AAGGGCCTGGAGGTCACCATCAC | 68 | -4.4(-14.4,-10.0) |
8 | 4903-4925 | AAGCCCAACCTCAGCTACATCAT | 58 | -4.2(-13.2,-9.0) |
9 | 3572-3594 | AAGCAGGAGACTTCCTTGAAGCC | 53 | -4.0(-12.1,-8.1) |
10 | 1161-1183 | AATGCCCTTTGACCTCATGGTGT | 53 | -3.9(-12.7,-8.8) |
11 | 4118-4140 | AAGATCAACTCACCTGTAATAAA | 37 | -3.8(-9.1,-5.3) |
12 | 4663-4685 | AAGGCCTGTGAGCCAGGAGTGGA | 68 | -3.8(-13.2,-9.4) |
13 | 2598-2620 | AATCCGAGCCGTTCTCTACAATT | 53 | -3.7(-10.9,-7.2) |
14 | 925-947 | AAGCGCATTCCGATTGAGGATGG | 53 | -3.6(-12.5,-8.9) |
15 | 2848-2870 | AAGGTCGTGCCGGAAGGAATCAG | 63 | -3.5(-11.4,-7.9) |
16 | 2770-2792 | AAGACCGGCCTGCAGGAAGTGGA | 68 | -3.4(-11.4,-8.0) |
17 | 4843-4865 | AAGCTGGAGGAGAAGAAACACTA | 53 | -3.4(-12.1,-8.7) |
18 | 2097-2119 | AATGGACAAAGTCGGCAAGTACC | 47 | -3.4(-10.6,-7.2) |
19 | 4549-4571 | AAGGAGGATGGAAAGCTGAACAA | 53 | -3.3(-12.1,-8.8) |
20 | 4183-4205 | AAGAGGCCTCAGGATGCCAAGAA | 63 | -3.3(-12.3,-9.0) |
21 | 337-359 | AACAGGGAGTTCAAGTCAGAAAA | 47 | -3.2(-11.3,-8.1) |
22 | 1135-1157 | AAGACACCCAAGTACTTCAAACC | 42 | -3.2(-10.1,-6.9) |
23 | 673-695 | AAGATCCGAGCCTACTATGAAAA | 47 | -3.2(-10.3,-7.1) |
24 | 3890-3912 | AAGCCTTGGCTCAATACCAAAAG | 47 | -3.1(-10.9,-7.8) |
25 | 4570-4592 | AAGCTCTGCCGTGATGAACTGTG | 58 | -3.1(-11.1,-8.0) |
表3b.25聚体siRNA正义链序列:
1:138 | CGUGAUCCUUGGAGCCUCAAAUAUA |
2:212 | CAGAAUCUAGAUAUCUUGCUCAGAU |
3:229 | GCUCAGAUUGGUGACUCCGUCUCAU |
4:299 | GAACCCAGAUAGAUAGUCCACUGAA |
5:439 | GGAAUCCAGGUGGAGAUCUACUCUU |
6:645 | CAAGAGUUUGGAAGUAACCUUUACU |
7:740 | UGCCCACAGUAAGGCAGGCUGUAAA |
8:1046 | AAGCAUUCCCUAGAGAUCCAGAAAU |
9:1687 | GAAGGAGACACUGUCAUCAUCUCUU |
10:2106 | GCAAAUCCUUGAUACUGCUCAUCAU |
表4:
靶向人IFN-γ(登录号NM_000619)的siRNA序列:
表4a.19聚体siRNA
1:14 | UCAUCUGAAGAUCAGCUAU |
2:56 | CCUUUGGACCUGAUCAGCU |
3:477 | GCUGACUAAUUAUUCGGUA |
4:510 | CCAACGCAAAGCAAUACAU |
5:616 | GCAUCCCAGUAAUGGUUGU |
6:912 | UCCCAUGGGUUGUGUGUUU |
7:914 | CCAUGGGUUGUGUGUUUAU |
8:1007 | GCAAUCUGAGCCAGUGCUU |
9:1016 | GCCAGUGCUUUAAUGGCAU |
10:1106 | GCUUCCAAAUAUUGUUGAC |
表4b.25聚体siRNA正义链序列:
1:12 | GAUCAUCUGAAGAUCAGCUAUUAGA |
2:47 | CAGUUAAGUCCUUUGGACCUGAUCA |
3:494 | UAACUGACUUGAAUGUCCAACGCAA |
4:604 | CGAGGUCGAAGAGCAUCCCAGUAAU |
5:622 | CAGUAAUGGUUGUCCUGCCUGCAAU |
6:626 | AAUGGUUGUCCUGCCUGCAAUAUUU |
7:849 | GCAAGGCUAUGUGAUUACAAGGCUU |
8:907 | CAAGAUCCCAUGGGUUGUGUGUUUA |
9:918 | GGGUUGUGUGUUUGAUUUCACUUGAU |
10:1004 | CCUGCAAUCUGAGCCAGUGCUUUAA |
表5:
靶向人IL-1(登录号:NM_033292)的siRNA序列:
表5a.19聚体siRNA正义链序列:
1:767 | GCAAGUCCCAGAUAUACUA |
2:826 | GCCCAAGUUUGAAGGACAA |
3:827 | CCCAAGUUUGAAGGACAAA |
4:885 | CCUGGUGUGGUGUGGUUUA |
5:909 | UCAGUAGGAGUUUCUGGAA |
6:915 | GGAGUUUCUGGAAACCUAU |
7:924 | GGAAACCUAUCUUUACCAA |
8:1180 | CCACUGAAAGAGUGACUUU |
9:1270 | GAAGAGAUCCUUCUGUAAA |
10:1296 | GGAAUUAUGUCUGCUGAAU |
表5b.25聚体siRNA正义链序列:
1:769 | AAGUCCCAGAUAUACUACAACUCAA |
2:826 | GCCCAAGUUUGAAGGACAAACCGAA |
3:881 | CAGCCCUGGUGUGGUGUGGUUUAAA |
4:884 | CCCUGGUGUGGUGUGGUUUAAAGAU |
5:887 | UGGUGUGGUGUGGUUUAAAGAUUCA |
6:909 | UCAGUAGGAGUUUCUGGAAACCUAU |
7:913 | UAGGAGUUUCUGGAAACCUAUCUUU |
8:914 | AGGAGUUUCUGGAAACCUAUCUUUA |
9:1176 | CCCACCACUGAAAGAGUGACUUUGA |
10:1178 | CACCACUGAAAGAGUGACUUUGACA |
表6:
靶向人IL-6(登录号:NM_000600)的siRNA序列:
表6a.19聚体siRNA正义链序列:
1:250 | GCAUCUCAGCCCUGAGAAA |
2:258 | GCCCUGAGAAAGGAGACAU |
3:360 | GGAUGCUUCCAAUCUGGAU |
4:364 | GCUUCCAAUCUGGAUUCAA |
5:375 | GGAUUCAAUGAGGAGACUU |
6:620 | GCAGGACAUGACAACUCAU |
7:706 | GGCACCUCAGAUUGUUGUU |
8:710 | CCUCAGAUUGUUGUUGUUA |
9:768 | GCACAGAACUUAUGUUGUU |
10:949 | GGAAAGUGGCUAUGCAGUU |
表6b.25聚体siRNA正义链序列:
1:256 | CAGCCCUGAGAAAGGAGACAUGUAA |
2:359 | UGGAUGCUUCCAAUCUGGAUUCAAU |
3:429 | GAGGUAUACCUAGAGUACCUCCAGA |
4:446 | CCUCCAGAACAGAUUUGAGAGUAGU |
5:631 | CAACUCAUCUCAUUCUGCGCAGCUU |
6:705 | GGGCACCUCAGAUUGUUGUUGUUAA |
7:762 | CACUGGGCACAGAACUUAUGUUGUU |
8:767 | GGCACAGAACUUAUGUUGUUCUCUA |
9:768 | GCACAGAACUUAUGUUGUUCUCUAU |
10:1002 | UGGAAAGUGUAGGCUUACCUCAAAU |
表7:
靶向IL-8(登录号:NM_000584)的siRNA序列:
表7a.19聚体siRNA正义链序列:
1:1342 | ACUCCCAGUCUUGUCAUUG |
2:1345 | CCCAGUCUUGUCAUUGCCA |
3:1346 | CCAGUCUUGUCAUUGCCAG |
4:1364 | GCUGUGUUGGUAGUGCUGU |
5:1372 | GGUAGUGCUGUGUUGAAUU |
6:1373 | GUAGUGCUGUGUUGAAUUA |
7:1378 | GCUGUGUUGAAUUACGGAA |
8:1379 | CUGUGUUGAAUUACGGAAU |
9:1427 | ACUCCACAGUCAAUAUUAG |
表7a.25聚体siRNA正义链序列:
1:1364 | GCUGUGUUGGUAGUGCUGUGUUGAA |
2:1366 | UGUGUUGGUAGUGCUGUGUUGAAUU |
3:1372 | GGUAGUGCUGUGUUGAAUUACGGAA |
4:1374 | UAGUGCUGUGUUGAAUUACGGAAUA |
5:1375 | AGUGCUGUGUUGAAUUACGGAAUAA |
6:1378 | GCUGUGUUGAAUUACGGAAUAAUGA |
表8:
靶向TNF-α(登录号:NM_004862)的siRNA序列:
表8a.19聚体siRNA正义链序列:
1:163 | GGACACCAUGAGCACUGAA |
2:168 | CCAUGAGCACUGAAAGCAU |
3:430 | GCCUGUAGCCCAUGUUGUA |
4:516 | GCGUGGAGCUGAGAGAUAA |
5:811 | GCCCGACUAUCUCGACUUU |
6:993 | CCCAAGCUUAGAACUUUAA |
7:1072 | GCUGGCAACCACUAAGAAU |
8:1076 | GCAACCACUAAGAAUUCAA |
9:1301 | GCCAGCUCCCUCUAUUUAU |
10:1305 | GCUCCCUCUAUUUAUGUUU |
表8b.25聚体siRNA正义链序列:
1:906 | UGGAGUCGUGCAUAGGACUUGCAAA |
2:1002 | GAUCAUUGCCCUAUCCGAAUAUCUU |
3:1010 | CCCUAUCCGAAUAUCUUCCUGUGAU |
4:1146 | GAACCAGCCUUUAGUGCCUACCAUU |
5:1150 | CAGCCUUUAGUGCCUACCAUUAUCU |
6:1153 | CCUUUAGUGCCUACCAUUAUCUUAU |
7:1199 | GACAAAGAUCUUGCCUUACAGACUU |
8:1241 | GAUUCUGUAACUGCAGACUUCAUUA |
9:1244 | UCUGUAACUGCAGACUUCAUUAGCA |
10:1254 | CAGACUUCAUUAGCACACAGAUUCA |
表9:
靶向人CD80(登录号:NM_005191)的siRNA序列:
表9a.19聚体siRNA正义链序列:
1:398 | CCAAGUGUCCAUACCUCAA |
2:442 | GGUCUUUCUCACUUCUGUU |
3:504 | GCUGUCCUGUGGUCACAAU |
4:696 | GGGCACAUACGAGUGUGUU |
5:781 | GCUGACUUCCCUACACCUA |
6: 965 | GCAGCAAACUGGAUUUCAA |
7:1378 | GCUUUGCAGGAAGUGUCUA |
8:1652 | GCUGCUGGAAGUAGAAUUU |
9:1658 | GGAAGUAGAAUUUGUCCAA |
10:1682 | GGUCAACUUCAGAGACUAU |
表9b.25聚体siRNA正义链序列:
1:535 | GAGCUGGCACAAACUCGCAUCUACU |
2:599 | GGGACAUGAAUAUAUGGCCCGAGUA |
3:631 | CGGACCAUCUUUGAUAUCACUAAUA |
4:698 | GCACAUACGAGUGUGUUGUUCUGAA |
5:898 | GGAGAAGAAUUAAAUGCCAUCAACA |
6:1205 | GAAGGGAAAGUGUACGCCCUGUAUA |
7:1275 | CCUCCAUUUGCAAUUGACCUCUUCU |
8:1302 | GAACUUCCUCAGAUGGACAAGAUUA |
9:1565 | CAGAUUUCCUAACUCUGGUGCUCUU |
10:1766 | AGGAAGUAUGGCAUGAACAUCUUUA |
表10:
靶向人CD86(登录号:NM_175862)的siRNA序列:
表10a.19聚体siRNA正义链序列:
1:36 | GCUGCUGUAACAGGGACUA |
2:130 | GCACUAUGGGACUGAGUAA |
3:189 | CCUCUGAAGAUUCAAGCUU |
4:398 | CCUGAGACUUCACAAUCUU |
5:425 | GGACAAGGGCUUGUAUCAA |
6:466 | CCACAGGAAUGAUUCGCAU |
7:586 | GCUCAUCUAUACACGGUUA |
8:867 | GCUGUACUUCCAACAGUUA |
9:942 | CCUCGCAACUCUUAUAAAU |
10:1284 | CCAAGAGGAGACUUUAAUU |
表10b.25聚体siRNA正义链序列:
1:3 | AAGGCUUGCACAGGGUGAAAGCUUU |
2:315 | GAGGUAUACUUAGGCAAAGAGAAAU |
3:326 | AGGCAAAGAGAAAUUUGACAGUGUU |
4:479 | UCGCAUCCACCAGAUGAAUUCUGAA |
5:747 | ACGAGCAAUAUGACCAUCUUCUGUA |
6:760 | CCAUCUUCUGUAUUCUGGAAACUGA |
7:848 | CCACAUUCCUUGGAUUACAGCUGUA |
8:860 | GAUUACAGCUGUACUUCCAACAGUU |
9:1019 | CCAUAUACCUGAAAGAUCUGAUGAA |
10:1278 | CGUAUGCCAAGAGGAGACUUUAAUU |
表11:
靶向人MHC-II(登录号:NM_002119)的siRNA序列:
表11a.19聚体siRNA正义链序列:
1:2474 | GGCUCUGGAUGACUCUGAU |
2:2593 | GGUGGACUAGGAAGGCUUU |
3:2641 | GCCAAUCAAGGUACAAGUA |
4:2642 | CCAAUCAAGGUACAAGUAA |
5:2740 | GGGCUUCUUAAGAGAGAAU |
6:2790 | GGAAGUGGAGGAGAAUCAU |
7:2799 | GGAGAAUCAUCUCAGGCAA |
8:3149 | CCUAGUCACAGCUUUAAAU |
9:3233 | GCAGGAAUCAAGAUCUCAA |
10:3416 | GGAAAGGUGUUUCUCUCAU |
表11b.25聚体siRNA正义链序列:
1:2591 | GAGGUGGACUAGGAAGGCUUUCUGA |
2:2607 | GCUUUCUGAAGAACCUGGGUCUGUU |
3:2739 | UGGGCUUCUUAAGAGAGAAUAAGUU |
4:2843 | CCCUCUUUGUGUGAUCACAUGCAAA |
5:3092 | CCGACAGCUCCUGAGUUUAUAUCAU |
6:3097 | AGCUCCUGAGUUUAUAUCAUCUCAA |
7:3140 | GCUGUGUCUCCUAGUCACAGCUUUA |
8:3215 | CAGCCCUGUGUAGUUAGAGCAGGAA |
9:3389 | GCUUAGACGUUAACUUGAUGCAUCA |
10:3395 | ACGUUAACUUGAUGCAUCAUUGGAA |
表12:
靶向人MHC-I(登录号:NM_005516)的siRNA序列:
表12a.19聚体siRNA正义链序列:
1:29 | GGCUGGGAUCAUGGUAGAU |
2:33 | GGGAUCAUGGUAGAUGGAA |
3:106 | CCCACUCCUUGAAGUAUUU |
4:163 | GCUUCAUCUCUGUGGGCUA |
5:436 | GGUAUGAACAGUUCGCCUA |
6:464 | GGAUUAUCUCACCCUGAAU |
7:573 | GCCUACCUGGAAGACACAU |
8:863 | GCAGAGAUACACGUGCCAU |
9:980 | CCUUGGAUCUGUGGUCUCU |
10:1296 | CCACCUCUGUGUCUACCAU |
表12b.25聚体siRNA正义链序列:
1:100 | CGGGCUCCCACUCCUUGAAGUAUUU |
2:108 | CACUCCUUGAAGUAUUUCCACACUU |
3:457 | ACGGCAAGGAUUAUCUCACCCUGAA |
4:458 | CGGCAAGGAUUAUCUCACCCUGAAU |
5:868 | GAUACACGUGCCAUGUGCAGCAUGA |
6:998 | UGGAGCUGUGGUUGCUGCUGUGAUA |
7:1002 | GCUGUGGUUGCUGCUGUGAUAUGGA |
8:1266 | UAGCACAAUGUGAGGAGGUAGAGAA |
9:1282 | GGUAGAGAAACAGUCCACCUCUGUG |
10:1286 | GAGAAACAGUCCACCUCUGUGUCUA |
表13:
靶向人CD28(登录号:NM_006139)的siRNA序列:
表13a.19聚体siRNA正义链序列:
1:69 | CCUUGAUCAUGUGCCCUAA |
2:234 | GCUCUUGGCUCUCAACUUA |
3:241 | GCUCUCAACUUAUUCCCUU |
4:306 | GCUUGUAGCGUACGACAAU |
5:494 | GCAAUGAAUCAGUGACAUU |
6:631 | GGGAAACACCUUUGUCCAA |
7:726 | GCUAGUAACAGUGGCCUUU |
8:830 | GCAAGCAUUACCAGCCCUA |
9:1216 | GCACAUCUCAGUCAAGCAA |
10:1413 | CCACGUAGUUCCUAUUUAA |
表13b.25聚体siRNA正义链序列:
1:53 | CCUUGUGGUUUGAGUGCCUUGAUCA |
2:228 | CAGGCUGCUCUUGGCUCUCAACUUA |
3:229 | AGGCUGCUCUUGGCUCUCAACUUAU |
4:325 | GCGGUCAACCUUAGCUGCAAGUAUU |
5:503 | CAGUGACAUUCUACCUCCAGAAUUU |
6:605 | GCAAUGGAACCAUUAUCCAUGUGAA |
7:1351 | GGGAGGGAUAGGAAGACAUAUUUAA |
8:1407 | AAUGAGCCACGUAGUUCCUAUUUAA |
9:1577 | UCCCUGUCAUGAGACUUCAGUGUUA |
10:1584 | CAUGAGACUUCAGUGUUAAUGUUCA |
表14:
靶向人CTLA4(登录号:AF414120)的siRNA序列:
表14a.19聚体siRNA正义链序列:
1:33 | GGGAUCAAAGCUAUCUAUA |
2:58 | CCUUGAUUCUGUGUGGGUU |
3:62 | GAUUCUGUGUGGGUUCAAA |
4:154 | CCAUGGCUUGCCUUGGAUU |
5:316 | CCAGCUUUGUGUGUGAGUA |
6:538 | UCUGCAAGGUGGAGCUCAU |
7:566 | GCCAUACUACCUGGGCAUA |
8:585 | GGCAACGGAACCCAGAUUU |
9:586 | GCAACGGAACCCAGAUUUA |
10:591 | GGAACCCAGAUUUAUGUAA |
表14b.25聚体siRNA正义链序列:
1:26 | CAUAUCUGGGAUCAAAGCUAUCUAU |
2:147 | CAUAAAGCCAUGGCUUGCCUUGGAU |
3:314 | CGCCAGCUUUGUGUGUGAGUAUGCA |
4:402 | GAAGUCUGUGCGGCAACCUACAUGA |
5:430 | GGAAUGAGUUGACCUUCCUAGAUGA |
6:441 | ACCUUCCUAGAUGAUUCCAUCUGCA |
7:581 | CAUAGGCAACGGAACCCAGAUUUAU |
8:587 | CAACGGAACCCAGAUUUAUGUAAUU |
9:590 | CGGAACCCAGAUUUAUGUAAUUGAU |
10:644 | CCUCUGGAUCCUUGCAGCAGUUAGU |
表15:
靶向人细小病毒B19(登录号:AY903437)的siRNA序列:
表15a.19聚体siRNA正义链序列:
1:398 | CCAAGUGUCCAUACCUCAA |
2:442 | GGUCUUUCUCACUUCUGUU |
3:504 | GCUGUCCUGUGGUCACAAU |
4:696 | GGGCACAUACGAGUGUGUU |
5:781 | GCUGACUUCCCUACACCUA |
6:965 | GCAGCAAACUGGAUUUCAA |
7:1378 | GCUUUGCAGGAAGUGUCUA |
8:1652 | GCUGCUGGAAGUAGAAUUU |
9:1658 | GGAAGUAGAAUUUGUCCAA |
10:1682 | GGUCAACUUCAGAGACUAU |
表15b.25聚体siRNA正义链序列:
1:729 | ACAGUGUGUGUAGAAGGCUUGUUUA |
2:807 | GGAAUGACUACUAAGGGAAAGUAUU |
3:1679 | CAGCAACGGUGACAUUACCUUUGUU |
4:1749 | GAGCGAAUGGUAAAGCUAAACUUUA |
5:2230 | UGCCUGUUUGUUGUGUGCAGCAUAU |
6:2360 | UAGCUGCCAUGUCGGAGCUUCUAAU |
7:2622 | CCUGUUUGACUUAGUUGCUCGUAUU |
8:3474 | CCCUGAUGCUUUAACUGUUACCAUA |
9:4083 | UGGCACUAGUCAAAGUACCAGAAUA |
10:4470 | GGGUUUACAUCAACCACCUCCUCAA |
在一个实施方案中,25个碱基对,具有平末端的siRNA双链体在体外和体内都显示出比19个碱基对,在两个3′末端都具有突出端更有效的基因敲减功效。
在本发明的另外的方面提供了双链多核苷酸,其包括如上所述的第一线性多核苷酸链,和与第一链的至少第一核苷酸序列互补并与其杂交形成双链siRNA组合物的第二多核苷酸链。
制剂
使用各种载体来制备含有siRNA的制剂或药物组合物。在几个实施方案中,本发明的siRNA多核苷酸通过脂质体介导的转染被递送至培养的细胞或等待移植的器官的细胞中,例如通过使用市场上可买到的试剂或技术,例如OligofectamineTM、LipofectAmineTM试剂、LipofectAmine 2000TM(Invitrogen),以及通过电穿孔,和相似技术。
含有siRNA的药物组合物包括保护siRNA稳定性、延长siRNA寿命、增强siRNA功能或使siRNA靶向特定组织/细胞的附加组分。这些包括各种可生物降解的聚合物、阳离子聚合物(例如聚乙烯亚胺)、阳离子共聚物例如组氨酸-赖氨酸(HK)多肽,参见例如PCT出版物Mixson等的WO01/47496、 Bio聚体ieux的WO 02/096941和麻省理工学院的WO 99/42091、聚乙二醇化的阳离子多肽和配体掺入聚合物等带正电荷的多肽、PolyTran溶液(HK聚合物和多糖的盐水或水溶液,多糖例如天然多糖,又名硬葡聚糖)、TargeTran(由包括靶向配体的缀合RGD-PEG-PEI聚合物组成的纳米颗粒的盐水或水悬液)、表面活性剂(Infasurf;Forest Laboratories,Inc.;ONY Inc.),和阳离子聚合物(例如聚乙烯亚胺)。(calfactant)是分离自小牛肺的用于气管内灌注的天然肺表面活性剂;它含有磷脂、天然脂类和疏水表面活性剂缔合的蛋白B和C。
这些聚合物可以是单维或多维的,也可以是直径小于20微米、20至100微米、或100微米以上的微粒或纳米颗粒。所述聚合物可以携带对于特定组织或细胞的受体或分子特异性的配体分子,因此用于siRNA的定向递送。siRNA多核苷酸也可以通过基于阳离子脂质体的载体,例如DOTAP、DOTAP/Cholesterol(Qbiogene,Inc.)和其他类型的脂质水溶液来递送。此外,低百分比(5-10%)葡萄糖水溶液,和Infasurf是空运递送siRNA的有效载体(Li B.J.et al,2005,Nature Medicine,11,944-951)。
此外,载体可以包括高渗枸橼酸盐溶液(560mOsm/kg氢氯化葡甲胺,560mOsm/kg碘克酸葡甲胺和600mOsm/kg碘克酸钠等等)。威斯康星大学溶液具有增加和延长心脏、肾、肺和肝保藏的潜能。威斯康星大学溶液广泛用于冷藏和运输指定为胰岛分离物的人供体胰。
该组合物可以进一步包含聚合载体。该聚合载体可以包括与RNA分子结合的阳离子聚合物。该阳离子聚合物可以是氨基酸共聚物,包括例如组氨酸和赖氨酸残基。该聚合物可以包括分支聚合物。
该组合物可以包含寻靶合成载体。该合成载体可以包括阳离子聚合物、亲水聚合物和靶向配体。该聚合物可以包括聚乙烯亚胺,亲水聚合物可以包括聚乙二醇或聚缩醛,和靶向配体可以包括包含RGD序列的肽。
siRNA/载体可以以非特异性方式配制为贮藏溶液或灌注介质,或配制为经体循环的靶向递送系统。
改善实体器官和细胞的移植
本发明提供了预防实体器官移植中的同种异体移植排斥和缺血/再灌注损伤的方法,是通过导入RNA干扰(siRNA)来沉默或下调靶基因表达。在本发明的方法中,siRNA以器官贮藏溶液的形式用于意欲移植的器官,即从供体取出后和当正在运输给受体时。移植器官、组织和/或细胞的供体或受体可以是哺乳动物,包括但不限于人、非人哺乳动物、非人灵长类动物、大鼠、小鼠、猪、狗、牛和马。指定用于移植的器官由包含siRNA寡核苷酸或多个siRNA寡核苷酸作为混合物的器官贮藏溶液维持。siRNA可以容易地和选择性到达供体器官和细胞,这促进潜在有害的全身副作用的降低。
在目前的实践中,供体器官经受冲洗并贮藏在静态或再循环系统,处于低体温条件(对于人,低于37℃,例如4℃)或正常热力条件下(对于人是37℃),处于特别配制的溶液(器官保藏溶液)中,为了洗去杂质和降低运输过程中的损害。本发明的方法包括器官保藏过程中供体器官和细胞的siRNA转染。这是一种有吸引力的方法,因为siRNA离体施加给待捐献的器官不会全身给予器官受体,且治疗可以专门递送至炎症部位。这种方法可用于预防移植失败,而无全身副作用。
siRNA转染制剂用于在原位和/或离体,或静态或机械灌注器官贮藏器中冲洗实体供体器官。配制的溶液具有局部注射给实体器官和通过将其浸没在siRNA制剂中而浸泡整个实体器官的用途。
siRNA试剂可以作为单个或多个双链体使用,靶向单个或多个基因,转染或不转染载体来处理移植器官(组织)和细胞。转染试剂包括但不限于合成聚合物、脂质体和糖等等。siRNA试剂还可以和其他试剂一起使用,例如小分子和单克隆抗体抑制剂、免疫调节剂和其他类型寡核苷酸。用siRNA/载体溶液注射和浸没用于移植的器官会将组织损伤和宿主排斥降至最低,和因此将增加移植器官在器官功能和存活率和使伴随发病率最小化方面的成功。
也在本发明中,在移植过程中可以用siRNA/载体制剂处理各种器官和细胞。所有实体器官移植基本上都需要手术制备供体,可以包括身体或待用于移植的具体器官的冲洗灌注。灌注可以用一种或多种流体。取出器官,在运输给受体过程中贮藏,和器官手术移植给受体。对本发明方法有用的器官包括但不限于肾、肝、心脏、胰、胰岛、小肠、肺、角膜、肢体和皮肤,以及这些器官中每一种器官相应的培养细胞。一个实例,肝细胞系开始发展为分离的肝细胞移植的万能供体,这是比用于治疗代谢性肝疾病的常位肝移植创伤小的方法。经由途径例如CD28/B7或CD40/CD40L共同刺激是这种移植成功的主要忧虑(2)。因此,使用siRNA/载体制剂沉默CD28或CD40途径将是改善移植成功率的优良策略。
另一个肾移植失败的实例是实体器官移植后细小病毒B19(PV-B19)的感染,其可以引起纯红细胞再生障碍性贫血(PRCA)。免疫抑制的移植受体的PV-B19感染合并严重的发病(1)。使用siRNA抑制PV-B19或任何其他病毒感染和复制是通过在移植初期期间处理供体器官和移植受体,从而改善肾移植的辅助疗法。
在另一个方面,本发明提供了包含一个或多个siRNA双链体的组合物,其中siRNA可以同时靶向涉及异体移植物或异种移植排斥或缺血/再灌注损伤的几个基因。多个siRNA双链组合可以更有效用于移植同种异体移植排斥或缺血/再灌注损伤。
免疫调节过程提供了使用本发明方法进行siRNA沉默的过多分子靶,例如(1)与激活相关的淋巴细胞上的分子;(2)刺激淋巴细胞的抗原呈递细胞(APCs)上的分子,例如II型MHC和共刺激分子;(3)可溶分子信号,例如细胞因子,例如TNF-α、IFN-β、IL-1、IL-6、IL-8;(4)与淋巴细胞渗出和回复原位相关的分子,例如血管细胞粘附分子-1、细胞间粘附分子-1;和(5)免疫效应分子,例如但不限于补体因子C3。另外候选靶基因包括细胞间粘附分子-1、I型主要组织相容性复合体、II型主要组织相容性复合体、IFN-γ、CD80、CD86、CD40和CD40L。
本发明还提供了方法和组合物,其使用siRNA寡核苷酸混合物(siRNA-OC)作为本发明方法中有用的治疗剂或达到更强的抗血管生成功效,用于治疗癌症和炎症。siRNA寡核苷酸混合物包含靶向至少三个mRNA靶的至少三个双链体。siRNA寡核苷酸混合物可以包含表1-15所列的任何siRNA序列。在一个实施方案中,siRNA寡核苷酸混合物包含补体C3、MHC-II和IFNγ特异性的siRNA。本发明基于两个重要方面:第一,siRNA双链体是很强效的基因表达抑制剂,和每一个siRNA分子由具有相同化学性能的短的双链RNA寡核苷酸组成(21-23nt,或24-25nt,或26-29nt);第二,异体移植物或异种移植排斥和缺血/再灌注损伤部分地与内源基因过表达相关。因此,使用靶向多个基因的siRNA-OC代表了有利的治疗方法,这是由于siRNA双链体的化学均一性和下调引起多种疾病或损伤的基因的协同效应。本发明定义siRNA-OC是靶向至少三个基因的siRNA双链体的组合,这些双链体以各种比例,各种物理形式,并同时通过相同途径,或不同途径和时间施加给病变组织。
siRNA介导的沉默可以与靶向一个这种基因的单个siRNA,或靶向同一基因内几个靶序列,或靶向不同类型的各种基因的多个siRNA的组合(例如本段确定的)一起应用。例如,包含多个siRNA双链体的组合物可以以相同或不同比例存在。因此,在三个siRNA的混合物中,双链体I、双链体II和双链体III每个可以以每个总siRNA试剂的33.3%(w/w)存在,或作为非限制性实例,分别以20%、45%和35%存在。
除非另外定义,本文使用的所有技术和科学术语与本发明所属领域普通技术人员通常理解的含义相同。示例的方法和材料描述如下,尽管与本文描述的类似或等同的方法和材料可以用在本发明的实践或测试中。本文提及的所有出版物及其他参考文献的全部内容引入作为参考。在冲突的情况下,本说明书,包括定义将操纵。尽管本文引用了多个文件,这个引证不构成任何这些文件形成本领域公知常识的一部分的认同。贯穿本说明书和权利要求书,单词“包含”将被理解为暗示内含所述整数或整数组,但不排除任何其他整数或整数组。材料、方法和实施例仅是示例性的,不意欲限制。
实施例1:siRNA介导的C3体外表达敲减
RNA干扰阻断依照它们的序列的小独特区段的基因表达。可以利用这个自然过程减少特异性基因的转录。在移植中,经确认来自供体的补体C3迅速上调缺血/再灌注损伤(I/RI),有助于组织损伤。补体C3被称作各种形式损伤和免疫调节的局部介质,和是移植缺血/再灌注损伤后基因敲减的有效靶,该靶很可能也参与异体免疫调节。本研究设法开发siRNA下调供体器官中的C3基因表达。
用10μg/ml IL-1和0.1μg/ml IL-6刺激大鼠肾上皮细胞系以上调C3基因表达。刺激后72小时,用一板C3特异性siRNA之一转染细胞。
序列 i.d. | siRNA 序列 |
C3-1 | CTG GCT CAA CGA CGA AAG ATA |
C3-2 | CAC GGT AAG CAC CAA GAA GGA |
C3-3 | AAG GGT GGA ACT GTT GCA TAA |
48小时后,实时PCR测定C3表达。结果表明刺激后,未转染细胞中C3表达上调(图1)。siRNA处理的细胞与未用siRNA处理的对照细胞相比显示C3表达降低达到60%。这些实验从不特异性诱导IFNγ上调(siRNA的潜在非靶效应)的板中鉴定到最有效的C3 siRNA序列(在图1中标记为C3-3 siRNA)。
上述实验获得的候选C3 siRNA转染刺激的大鼠肾上皮细胞以表达C3,如上所述。这个C3特异性siRNA的浓度范围产生显著的(P<0.05)C3 mRNA敲减,如实时PCR测定(图2)。这个实验证明鉴定的C3 siRNA序列用于体内测试的技术可行性和功效。
实施例2:siRNA介导的C3体内表达敲减
然后如上述实验测定的最有效C3 siRNA包装入促进体内siRNA转染的合成聚阳离子纳米颗粒。纳米颗粒由PolyTran、分支组氨酸(H)和赖氨酸(K)聚合物家族组成,可有效体外、体内和离体传递siRNA。它们的核心序列如下:R-KR-KR-KR,其中R=[HHHKHHHKHHHKHHH]2KH4NH4。对于体内实验,初步测试下列分支HK聚合物将递送入siRNA异体移植物细胞的功效:H3K4b。这个分支聚合物具有如上所述的相同核心和结构,除了R分支不同:R=KHHHKHHHKHHHKHHHK。选择这些聚合物是因为它们对于不同形式核酸的体外或体内功效。分支HK聚合物溶于水溶液,然后以所列质量比与siRNA水溶液混合,形成直径平均粒度150-200nm的纳米颗粒。HKP-siRNA水溶液是半透明的,无引人注目的沉淀聚集体。这些溶液可以在4℃保存至少三个月。
向高渗枸橼酸盐灌注液体中添加纳米颗粒并给予供体大鼠肾。冷缺血4小时后,肾移植入同种同基因的宿主。两天后,收获肾并用实时PCR测定C3基因表达。非移植、未处理肾充当阴性对照(在图3中标记为NKC),而未用siRNA处理的灌注移植肾充当阳性对照(在图3中标记未ISCH)。以移植的未处理肾的mRNA水平对siRNA处理的肾的水平进行标准化。图3显示了结果。
结果证明C3-siRNA使移植后C3基因表达与未处理移植物相比降低62.56%(P<0.05,n=4),降至低于正常肾中检测到的水平。当与混杂FITC标记的siRNA对照相比,C3基因表达减少73.34%(P<0.05,n=4)。FITC标记的混杂siRNA对照比未处理肾显示出更强的C3基因表达下调,暗示非靶效应。组织学显示移植前用C3siRNA处理的肾免于缺血/再灌注损伤(I/RI)(图4),但是用FITC标记的混杂siRNA灌注的细胞和组织的直接荧光显微术显示组织中不含任何可检测到的siRNA。
总之,与对照相比,siRNA抑制C3基因表达有效地降低了局部C3活性。纳米颗粒策略看起来克服了有效siRNA递送的问题。现在看来可能开发系列特异性siRNA以降低供体器官中的促炎基因表达,作为常规免疫抑制或诱发耐受的辅助治疗。
实施例3:通过噬菌体展示确定集中于移植肾的肽序列
为了提供含siRNA的纳米颗粒的器官靶向特异性,可以通过噬菌体展示鉴定集中于感兴趣器官的肽。使用这个方法鉴定如上所述的肾移植大鼠模型的候选靶向肽。用高渗枸橼酸盐冲洗供体肾并在移植入同种同基因宿主前于4℃储存4小时。48小时后,麻醉受体并经尾静脉注射制备的半胱氨酸限制的7聚体噬菌体文库(New EnglandBiolabs)。5分钟后,收获移植肾和从肾取出噬菌体,第一轮“体内生物淘选”。在注射给另一个肾移植受体前,提取的噬菌体在大肠杆菌中扩充。重复这种生物淘选总共三轮。每轮后,取噬菌体样品,估计移植肾中存在的数量。每次扩充后,噬菌体样品以菌落生长在琼脂平板上,因此可以分离噬菌体并可以测定表达文库肽的DNA序列。图5(下图)显示了每轮生物淘选后,从移植肾中回收的噬菌体(随机噬菌体)与对照寻靶链霉抗生物素蛋白(R3vsStrep)相比增加的数量。鉴定的集中于肾的肽序列的实例是C-LPSPKRT-C、C-LPSPKKT-C、C-PTSVPKT-C。第三轮生物淘选后,噬菌体集中于移植肾,和在受体的其他器官中找到的数量低得多(图5,下图)。这些候选肽掺入TargeTran纳米颗粒以提供siRNA靶向移植器官的特异性。
参考文献
1.Subtirelu MM et al. Acute renal failure in apediatric kidney allograft recipient treated withintravenous immunoglobulin for parvovirus B19induced pure red cell aplasia.Pediatr Transplant.2005 Dec;9(6):801-4.
2.Sampietro R,et al. Extension of the adult hepaticallograft pool using split liver transplantation.Acta Gastroenterol Belg.2005 Jul-Sep;68(3):369-75.
3.Chalermskulrat W,et al.Combined donor-specifictransfusion and anti-CD154 therapy achieves airwayallograft tolerance.Thorax.2005 Oct 27;[Epubahead of print].
4.Oliveira JG,et al.Humoral immune response afterkidney transplantation is enhanced by acuterejection and urological obstruction and is down-regulated by mycophenolate mofetil treatment.Transpl Int.2005 Nov;18(11):1286-91.
5.McManus,M.T.and P.A.Sharp(2002)Gene silencingin mammals by small interfering RNAs.NatureReview,Genetics.3(10):737-747.
6.Lu,P.Y.et al.(2003)siRNA-mediatedantitumorigenesis for drug target validation andtherapeutics.Current Opinion in MolecularTherapeutics.5(3):225-234.
7.Lu,P.Y.et al(2002)Tumor inhibition by RNAi-mediated VEGF and VEGFR2 down regulation inxenograft models.Cancer Gene Therapy.10(Supplement))S4.
8.Kim,B.et al.(2004)Inhibition of ocularangiogenesis by siRNA targeting vascularendothelial growth factor-pathway genes;therapeutic strategy for herpetic stromalkeratitis.Am.J.Pathol.165(6):2177-85.
9.Lu,P.Y.and M.Woodle(2005)Delivering siRNA invivo For functional genomics can noveltherapeutics.In RNA Interference Technology.Cambridge University Press.P303-317.
10.Lu,P.Y.et al.(2005)Modulation of angiogenesiswith siRNA inhibitors for novel therapeutics.TRENDS in Molecular Medicine.11(3),104-13.
Claims (45)
1.靶向待捐献给受试者的器官的细胞中存在的免疫调节或免疫效应基因的寻靶多核苷酸。
2.根据权利要求1的寻靶多核苷酸,其中多核苷酸是单链线性多核苷酸、双链线性多核苷酸或发夹多核苷酸。
3.根据权利要求1的寻靶多核苷酸,其包含靶向选自表1-15公开序列的序列的第一核苷酸序列。
4.一种抑制移植器官被该器官的受体排斥的方法,包括在将器官移植给受体前,使其与包含根据权利要求1的寻靶多核苷酸的组合物接触的步骤。
5.根据权利要求4的方法,其中该组合物包含根据权利要求2的寻靶多核苷酸。
6.根据权利要求4的方法,其中该组合物包含根据权利要求3的寻靶多核苷酸。
7.根据权利要求4的方法,其中接触步骤包括用该组合物灌注该器官。
8.根据权利要求4的方法,其中接触步骤包括将该器官浸泡或浸没在该组合物中。
9.根据权利要求4的方法,其中该组合物包含多个根据权利要求1的寻靶多核苷酸。
10.根据权利要求4的方法,其中在移植给受试者前的器官贮藏期间,接触步骤有效下调免疫调节或免疫效应靶基因。
11.根据权利要求4的方法,其中多核苷酸抑制器官的细胞中的靶基因表达。
12.根据权利要求4的方法,其中所述器官是供体的器官。
13.根据权利要求4的方法,其中所述器官是肾。
14.根据权利要求4的方法,其中所述器官是肝。
15.根据权利要求4的方法,其中所述移植器官是肺。
16.根据权利要求4的方法,其中所述器官是胰。
17.根据权利要求4的方法,其中所述器官是心脏。
18.根据权利要求4的方法,其中所述器官是小肠。
19.根据权利要求4的方法,其中所述器官是角膜。
20.根据权利要求4的方法,其中该器官包含选自下组的细胞:上皮细胞、血管内皮、血管平滑肌细胞、心肌(心脏)和移植时器官中居留的过客白细胞。
21.根据权利要求4的方法,其中所述受体是人。
22.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表1所列序列的C3(补体C3)序列。
23.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表2所列序列的ICAM1(细胞间粘附分子-1)序列。
24.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表3所列序列的VCAM-1(血管细胞粘附分子-1)序列。
25.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表4所列序列的IFN-γ(干扰素γ)序列。
26.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表5所列序列的IL-1(白介素-1)序列。
27.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表6所列序列的IL-6(白介素-6)序列。
28.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表7中所列序列的IL-8(白介素-8)序列。
29.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表8中所列序列的TNF-α(肿瘤坏死因子-α)序列。
30.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表9中所列序列的CD80序列。
31.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表10中所列序列的CD86序列。
32.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表11中所列序列的MHC-II(II型主要组织相容性复合体)序列。
33.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表12中所列序列的MHC-I(I型主要组织相容性复合体)序列。
34.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表13中所列序列的CD28序列。
35.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表14中所列序列的CTLA-4序列。
36.根据权利要求6的方法,其中寻靶多核苷酸靶向选自表15中所列序列的PV-B19序列。
37.根据权利要求4的方法,其中组合物进一步包含灌注液体。
38.根据权利要求4的方法,其中组合物进一步包含Polytran聚合物溶液。
39.根据权利要求4的方法,其中组合物进一步包含TargeTran纳米颗粒溶液。
40.根据权利要求37的方法,其中灌注液体是高渗枸橼酸盐溶液或威斯康星大学溶液。
41.根据权利要求4的方法,其中所述寻靶多核苷酸包含针对一个或多个基因序列的一个或多个siRNA双链体。
42.根据权利要求4的方法,其中寻靶多核苷酸与小分子药物、单克隆抗体药物或其他免疫调节剂联合使用。
43.根据权利要求4的方法,其中组合物包含多个寻靶多核苷酸,并且其中多核苷酸靶向多个基因序列。
44.根据权利要求43的方法,其中寻靶多核苷酸是靶向C3、TNF-α和IL-8基因序列的混合物。
45.根据权利要求4的方法,其中组合物被进一步给予器官受体。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US74115705P | 2005-11-30 | 2005-11-30 | |
US60/741,157 | 2005-11-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN101426913A true CN101426913A (zh) | 2009-05-06 |
Family
ID=38092807
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNA2006800520917A Pending CN101426913A (zh) | 2005-11-30 | 2006-11-30 | 使用siRNA敲减基因表达和改善实体器官和细胞移植的组合物和方法 |
Country Status (6)
Country | Link |
---|---|
US (1) | US20100028848A1 (zh) |
EP (1) | EP1963508A2 (zh) |
JP (1) | JP2009518008A (zh) |
CN (1) | CN101426913A (zh) |
CA (1) | CA2670801A1 (zh) |
WO (1) | WO2007064846A2 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105664154A (zh) * | 2014-10-23 | 2016-06-15 | 江苏命码生物科技有限公司 | 降低组织和/或器官移植性免疫排斥的方法及其应用 |
WO2023186056A1 (zh) * | 2022-04-02 | 2023-10-05 | 上海舶望制药有限公司 | 用于抑制补体成分c3蛋白表达的组合物和方法 |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8361976B2 (en) | 2004-07-09 | 2013-01-29 | University Of Massachusetts | Therapeutic alteration of transplantable tissues through in situ or ex vivo exposure to RNA interference molecules |
JP2008520209A (ja) * | 2004-11-17 | 2008-06-19 | ユニヴァーシティ・オブ・メリーランド,バルチモア | siRNAの有効なキャリアとしての高度に枝分かれしたHKペプチド |
EP1966379B1 (en) * | 2005-12-22 | 2010-04-28 | OPKO Ophthalmics, LLC | Compositions and methods for regulating complement system |
US8318693B2 (en) | 2008-09-02 | 2012-11-27 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of mutant EGFR gene |
US8389474B1 (en) * | 2009-07-14 | 2013-03-05 | Alan Anson Wanderer | Rationale for IL-1 β targeted therapy to improve harvested organ viability, allograft tolerance, replant success and for conditions characterized by reduced or absent arterial perfusion |
DE102011118024A1 (de) * | 2011-08-01 | 2013-02-07 | Technische Universität Dresden | Inhibitor der Expression der Pro-Caspase 1 |
US10652525B2 (en) * | 2013-10-31 | 2020-05-12 | 3Di Llc | Quad view display system |
WO2015070080A2 (en) * | 2013-11-08 | 2015-05-14 | Dana-Farber Cancer Institute, Inc. | Nucleic acid nanostructures for in vivo agent delivery |
AU2014362262B2 (en) * | 2013-12-12 | 2021-05-13 | Alnylam Pharmaceuticals, Inc. | Complement component iRNA compositions and methods of use thereof |
US9994811B2 (en) * | 2014-10-09 | 2018-06-12 | Lauren Brasile | Reducing the immunogenicity of allografts |
US11414694B2 (en) | 2016-03-11 | 2022-08-16 | Children's Medical Center Corporation | Nucleic acid nanoswitch catenanes |
CN115948505A (zh) | 2016-08-02 | 2023-04-11 | 哈佛学院院长及董事 | 交叉协同自组装体 |
CN106636090B (zh) * | 2016-10-11 | 2019-08-09 | 上海优卡迪生物医药科技有限公司 | 人源白细胞介素6的siRNA、重组表达CAR-T载体及其构建方法和应用 |
JP2019535839A (ja) | 2016-11-29 | 2019-12-12 | ピュアテック ヘルス エルエルシー | 治療剤の送達のためのエクソソーム |
EP3406139A1 (en) | 2017-05-26 | 2018-11-28 | Medizinische Hochschule Hannover | Method for genetically modifying a vascularised tissue |
EP3704252A1 (en) * | 2017-11-01 | 2020-09-09 | Alnylam Pharmaceuticals, Inc. | Complement component c3 irna compositions and methods of use thereof |
WO2023076451A1 (en) | 2021-10-29 | 2023-05-04 | Alnylam Pharmaceuticals, Inc. | Complement factor b (cfb) irna compositions and methods of use thereof |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030096775A1 (en) * | 2001-10-23 | 2003-05-22 | Isis Pharmaceuticals Inc. | Antisense modulation of complement component C3 expression |
CA2388441A1 (en) * | 2002-06-10 | 2003-12-10 | Wei-Ping Min | Immunomodulation using rna interference |
DE112004002914A5 (de) * | 2004-05-06 | 2007-05-24 | Medizinische Hochschule Hannover | Verbindungen und Verfahren zur Immunsuppression |
US8361976B2 (en) * | 2004-07-09 | 2013-01-29 | University Of Massachusetts | Therapeutic alteration of transplantable tissues through in situ or ex vivo exposure to RNA interference molecules |
US20080311552A1 (en) * | 2005-09-20 | 2008-12-18 | London Health Sciences Centre Research, Inc. | Use of Sirnas in Organ Storage/Reperfusion Solutions |
-
2006
- 2006-11-30 JP JP2008543476A patent/JP2009518008A/ja not_active Withdrawn
- 2006-11-30 CN CNA2006800520917A patent/CN101426913A/zh active Pending
- 2006-11-30 EP EP06838740A patent/EP1963508A2/en not_active Withdrawn
- 2006-11-30 WO PCT/US2006/045933 patent/WO2007064846A2/en active Search and Examination
- 2006-11-30 US US12/085,873 patent/US20100028848A1/en not_active Abandoned
- 2006-11-30 CA CA002670801A patent/CA2670801A1/en not_active Abandoned
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105664154A (zh) * | 2014-10-23 | 2016-06-15 | 江苏命码生物科技有限公司 | 降低组织和/或器官移植性免疫排斥的方法及其应用 |
CN105664154B (zh) * | 2014-10-23 | 2021-02-12 | 江苏命码生物科技有限公司 | 降低组织和/或器官移植性免疫排斥的方法及其应用 |
WO2023186056A1 (zh) * | 2022-04-02 | 2023-10-05 | 上海舶望制药有限公司 | 用于抑制补体成分c3蛋白表达的组合物和方法 |
Also Published As
Publication number | Publication date |
---|---|
EP1963508A2 (en) | 2008-09-03 |
WO2007064846A2 (en) | 2007-06-07 |
CA2670801A1 (en) | 2007-06-07 |
JP2009518008A (ja) | 2009-05-07 |
US20100028848A1 (en) | 2010-02-04 |
WO2007064846A3 (en) | 2008-02-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101426913A (zh) | 使用siRNA敲减基因表达和改善实体器官和细胞移植的组合物和方法 | |
Riazifar et al. | Stem cell extracellular vesicles: extended messages of regeneration | |
US20220273566A1 (en) | Nanomaterials containing constrained lipids and uses thereof | |
Ichim et al. | RNA interference: a potent tool for gene-specific therapeutics | |
Robey et al. | Systems approaches to preventing transplanted cell death in cardiac repair | |
JP6364009B2 (ja) | P53に対する二本鎖オリゴヌクレオチド分子、およびその使用方法 | |
US6022863A (en) | Regulation of gene expression | |
CN100556461C (zh) | 抗Smad7的反义寡核苷酸(ODN)及其在医药领域中的用途 | |
US20060063731A1 (en) | In vivo inhibition of hepatitis B virus | |
JP2005519976A (ja) | 動物細胞への阻害剤の伝達によるrna機能の阻害 | |
JP2007527240A (ja) | アレルギー性鼻炎および喘息のためのRNAiベースの治療 | |
Chang et al. | Inhibition of keratin 17 expression with antisense and RNAi strategies: exploring novel therapy for psoriasis | |
Tesovnik et al. | Extracellular vesicles derived human-miRNAs modulate the immune system in type 1 diabetes | |
Velu et al. | Utilizing antagomiR (antisense microRNA) to knock down microRNA in murine bone marrow cells | |
CN111065732A (zh) | 肿瘤类器官模型 | |
CN103889433A (zh) | 使用脐带组织来源的细胞治疗外周血管疾病 | |
CN108431228A (zh) | Tlr抑制性寡核苷酸及其用途 | |
Li et al. | Simultaneous blockage of contextual TGF-β by cyto-pharmaceuticals to suppress breast cancer metastasis | |
CN102712920A (zh) | 缺氧调节的条件沉默性aav表达血管生成诱导因子 | |
Carlson et al. | Interleukin‐10 and Transforming Growth Factor‐β Cytokines Decrease Immune Activation During Normothermic Ex Vivo Machine Perfusion of the Rat Liver | |
CN109498652B (zh) | 诱导型调节性t细胞来源的外泌体的应用 | |
CN111388499A (zh) | miR-31在制备预防和治疗溃疡性结肠炎的结肠靶向纳米药物中的应用 | |
CN111107856A (zh) | 增强基于t细胞的免疫疗法的效力的组合物和方法 | |
WO1995003694A1 (en) | Method for making universal donor cells | |
Martins et al. | Organ therapeutics during ex-situ dynamic preservation. a look into the future |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1130288 Country of ref document: HK |
|
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20090506 |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: WD Ref document number: 1130288 Country of ref document: HK |