US20150031871A1 - RNA-Mediated Gene Modulation - Google Patents
RNA-Mediated Gene Modulation Download PDFInfo
- Publication number
- US20150031871A1 US20150031871A1 US14/084,512 US201314084512A US2015031871A1 US 20150031871 A1 US20150031871 A1 US 20150031871A1 US 201314084512 A US201314084512 A US 201314084512A US 2015031871 A1 US2015031871 A1 US 2015031871A1
- Authority
- US
- United States
- Prior art keywords
- rna
- gene
- sprnai
- cell
- intron
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 128
- 230000001404 mediated effect Effects 0.000 title abstract description 12
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 113
- 108020005067 RNA Splice Sites Proteins 0.000 claims description 13
- 239000000203 mixture Substances 0.000 abstract description 36
- 230000006870 function Effects 0.000 abstract description 29
- 238000000034 method Methods 0.000 abstract description 28
- 102000019034 Chemokines Human genes 0.000 abstract description 4
- 108010012236 Chemokines Proteins 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 74
- 238000001890 transfection Methods 0.000 description 28
- 230000014509 gene expression Effects 0.000 description 26
- 108020004414 DNA Proteins 0.000 description 25
- 239000004055 small Interfering RNA Substances 0.000 description 24
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 22
- 239000003795 chemical substances by application Substances 0.000 description 22
- 230000030279 gene silencing Effects 0.000 description 22
- 239000013598 vector Substances 0.000 description 20
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 18
- 108020004999 messenger RNA Proteins 0.000 description 17
- 238000012226 gene silencing method Methods 0.000 description 16
- 108020004459 Small interfering RNA Proteins 0.000 description 15
- 230000037440 gene silencing effect Effects 0.000 description 15
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 14
- 230000009368 gene silencing by RNA Effects 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 241001465754 Metazoa Species 0.000 description 13
- 108700011259 MicroRNAs Proteins 0.000 description 13
- 208000015181 infectious disease Diseases 0.000 description 13
- 239000002679 microRNA Substances 0.000 description 13
- 108091027967 Small hairpin RNA Proteins 0.000 description 12
- 230000000295 complement effect Effects 0.000 description 12
- 239000012634 fragment Substances 0.000 description 12
- 238000001727 in vivo Methods 0.000 description 12
- 238000000636 Northern blotting Methods 0.000 description 11
- 230000000692 anti-sense effect Effects 0.000 description 11
- 201000010099 disease Diseases 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- 241000725303 Human immunodeficiency virus Species 0.000 description 10
- 108010022222 Integrin beta1 Proteins 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 10
- 238000011282 treatment Methods 0.000 description 10
- 108020005544 Antisense RNA Proteins 0.000 description 9
- 108091032955 Bacterial small RNA Proteins 0.000 description 9
- 102000012355 Integrin beta1 Human genes 0.000 description 9
- 102000000588 Interleukin-2 Human genes 0.000 description 9
- 108010002350 Interleukin-2 Proteins 0.000 description 9
- 210000001744 T-lymphocyte Anatomy 0.000 description 9
- 230000015556 catabolic process Effects 0.000 description 9
- 238000010367 cloning Methods 0.000 description 9
- 239000002299 complementary DNA Substances 0.000 description 9
- 239000003184 complementary RNA Substances 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 9
- 238000006731 degradation reaction Methods 0.000 description 9
- 210000004185 liver Anatomy 0.000 description 9
- 125000003729 nucleotide group Chemical group 0.000 description 9
- -1 stRNA Proteins 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 230000003612 virological effect Effects 0.000 description 9
- 108091092195 Intron Proteins 0.000 description 8
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 8
- 230000001413 cellular effect Effects 0.000 description 8
- 230000007246 mechanism Effects 0.000 description 8
- 239000002773 nucleotide Substances 0.000 description 8
- 210000003491 skin Anatomy 0.000 description 8
- 108060000903 Beta-catenin Proteins 0.000 description 7
- 108700005077 Viral Genes Proteins 0.000 description 7
- 241000700605 Viruses Species 0.000 description 7
- 230000001537 neural effect Effects 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- 102000004196 processed proteins & peptides Human genes 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 238000012545 processing Methods 0.000 description 7
- 230000001177 retroviral effect Effects 0.000 description 7
- 108091092562 ribozyme Proteins 0.000 description 7
- 210000001324 spliceosome Anatomy 0.000 description 7
- 210000000130 stem cell Anatomy 0.000 description 7
- 238000002560 therapeutic procedure Methods 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 102000015735 Beta-catenin Human genes 0.000 description 6
- 241000287828 Gallus gallus Species 0.000 description 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 210000002257 embryonic structure Anatomy 0.000 description 6
- 239000013604 expression vector Substances 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000013642 negative control Substances 0.000 description 6
- 238000013518 transcription Methods 0.000 description 6
- 230000035897 transcription Effects 0.000 description 6
- 238000002255 vaccination Methods 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 208000030507 AIDS Diseases 0.000 description 5
- 208000031886 HIV Infections Diseases 0.000 description 5
- 230000001154 acute effect Effects 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 238000010208 microarray analysis Methods 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 230000002265 prevention Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 4
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 4
- 108700024394 Exon Proteins 0.000 description 4
- 102000014150 Interferons Human genes 0.000 description 4
- 108010050904 Interferons Proteins 0.000 description 4
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 239000011543 agarose gel Substances 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000001684 chronic effect Effects 0.000 description 4
- 108010030074 endodeoxyribonuclease MluI Proteins 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 230000002452 interceptive effect Effects 0.000 description 4
- 229940079322 interferon Drugs 0.000 description 4
- 230000007774 longterm Effects 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 230000014616 translation Effects 0.000 description 4
- 239000013603 viral vector Substances 0.000 description 4
- 241000701022 Cytomegalovirus Species 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 108700029229 Transcriptional Regulatory Elements Proteins 0.000 description 3
- 108020000999 Viral RNA Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000036436 anti-hiv Effects 0.000 description 3
- 239000003816 antisense DNA Substances 0.000 description 3
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000001415 gene therapy Methods 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- 210000003494 hepatocyte Anatomy 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000000644 propagated effect Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 229920002477 rna polymer Polymers 0.000 description 3
- 230000001743 silencing effect Effects 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 230000006648 viral gene expression Effects 0.000 description 3
- QAPSNMNOIOSXSQ-YNEHKIRRSA-N 1-[(2r,4s,5r)-4-[tert-butyl(dimethyl)silyl]oxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O[Si](C)(C)C(C)(C)C)C1 QAPSNMNOIOSXSQ-YNEHKIRRSA-N 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 241000014654 Adna Species 0.000 description 2
- 101150076489 B gene Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 241000746758 Heteractis crispa Species 0.000 description 2
- 101000907904 Homo sapiens Endoribonuclease Dicer Proteins 0.000 description 2
- 102000003814 Interleukin-10 Human genes 0.000 description 2
- 108090000174 Interleukin-10 Proteins 0.000 description 2
- 102000013691 Interleukin-17 Human genes 0.000 description 2
- 108050003558 Interleukin-17 Proteins 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 229930193140 Neomycin Natural products 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 208000010513 Stupor Diseases 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
- 101710204645 UV excision repair protein RAD23 homolog B Proteins 0.000 description 2
- 102100020779 UV excision repair protein RAD23 homolog B Human genes 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
- 238000009098 adjuvant therapy Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 239000000074 antisense oligonucleotide Substances 0.000 description 2
- 238000012230 antisense oligonucleotides Methods 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 102000005936 beta-Galactosidase Human genes 0.000 description 2
- 102000023732 binding proteins Human genes 0.000 description 2
- 108091008324 binding proteins Proteins 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 239000005547 deoxyribonucleotide Substances 0.000 description 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 238000001476 gene delivery Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 210000004209 hair Anatomy 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229940076144 interleukin-10 Drugs 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 239000011859 microparticle Substances 0.000 description 2
- 229960004927 neomycin Drugs 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 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 1
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 101000997963 Aequorea victoria Green fluorescent protein Proteins 0.000 description 1
- 239000012116 Alexa Fluor 680 Substances 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 101100523940 Arabidopsis thaliana RAD23A gene Proteins 0.000 description 1
- 101100523944 Arabidopsis thaliana RAD23B gene Proteins 0.000 description 1
- 101100194005 Arabidopsis thaliana RAD23C gene Proteins 0.000 description 1
- 101100194006 Arabidopsis thaliana RAD23D gene Proteins 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 101100086373 Dictyostelium discoideum rcbA gene Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 102100030011 Endoribonuclease Human genes 0.000 description 1
- 102100023387 Endoribonuclease Dicer Human genes 0.000 description 1
- 108010093099 Endoribonucleases Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 208000034454 F12-related hereditary angioedema with normal C1Inh Diseases 0.000 description 1
- RZSYLLSAWYUBPE-UHFFFAOYSA-L Fast green FCF Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC(O)=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 RZSYLLSAWYUBPE-UHFFFAOYSA-L 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 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
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101000717424 Homo sapiens UV excision repair protein RAD23 homolog B Proteins 0.000 description 1
- 241000714260 Human T-lymphotropic virus 1 Species 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 102000004083 Lymphotoxin-alpha Human genes 0.000 description 1
- 108090000542 Lymphotoxin-alpha Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108091027974 Mature messenger RNA Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 101000892448 Neisseria gonorrhoeae Type II restriction enzyme NgoMIV Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 241001504519 Papio ursinus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 108700001094 Plant Genes Proteins 0.000 description 1
- 108091081045 Preribosomal RNA Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 101150105148 RAD23 gene Proteins 0.000 description 1
- 108020004518 RNA Probes Proteins 0.000 description 1
- 239000003391 RNA probe Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 1
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 239000004182 Tylosin Substances 0.000 description 1
- 229930194936 Tylosin Natural products 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 239000011324 bead Substances 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
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- 229960000603 cefalotin Drugs 0.000 description 1
- XIURVHNZVLADCM-IUODEOHRSA-N cefalotin Chemical compound N([C@H]1[C@@H]2N(C1=O)C(=C(CS2)COC(=O)C)C(O)=O)C(=O)CC1=CC=CS1 XIURVHNZVLADCM-IUODEOHRSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 108091092328 cellular RNA Proteins 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 230000027394 endothelium development Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000008029 eradication Effects 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- 238000011124 ex vivo culture Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 230000003659 hair regrowth Effects 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 208000016861 hereditary angioedema type 3 Diseases 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000008099 melanin synthesis Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000003990 molecular pathway Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- YFCUZWYIPBUQBD-ZOWNYOTGSA-N n-[(3s)-7-amino-1-chloro-2-oxoheptan-3-yl]-4-methylbenzenesulfonamide;hydron;chloride Chemical compound Cl.CC1=CC=C(S(=O)(=O)N[C@@H](CCCCN)C(=O)CCl)C=C1 YFCUZWYIPBUQBD-ZOWNYOTGSA-N 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 230000037311 normal skin Effects 0.000 description 1
- 230000020520 nucleotide-excision repair Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000005868 ontogenesis Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 210000004986 primary T-cell Anatomy 0.000 description 1
- 230000001566 pro-viral effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 239000000985 reactive dye Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 108020004418 ribosomal RNA Proteins 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 229960002385 streptomycin sulfate Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 229960004059 tylosin Drugs 0.000 description 1
- WBPYTXDJUQJLPQ-VMXQISHHSA-N tylosin Chemical compound O([C@@H]1[C@@H](C)O[C@H]([C@@H]([C@H]1N(C)C)O)O[C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CC=O)CO[C@H]1[C@@H]([C@H](OC)[C@H](O)[C@@H](C)O1)OC)CC)[C@H]1C[C@@](C)(O)[C@@H](O)[C@H](C)O1 WBPYTXDJUQJLPQ-VMXQISHHSA-N 0.000 description 1
- 235000019375 tylosin Nutrition 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 230000029812 viral genome replication 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
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
Definitions
- This invention relates to regulation of a gene function.
- One strategy for treating human diseases is to target specific disease-associated genes by either replacing impaired gene functions or by suppressing unwanted gene functions.
- Expression vectors are commonly used for introducing active genes into a cell to provide missing gene functions.
- antisense oligonucleotides, antibodies, and small molecule drugs are often used as therapeutic agents.
- RNA interference (Elbashir et al. (2001) Nature 411: 494-498) and deoxyribonucleotidylated-RNA interfering (D-RNAi) (Lin et al. (2001) Biochem. Biophys. Res. Commun. 281: 639-644) technologies in treating human diseases are also in progress.
- RNAi elicits post-transcriptional gene silencing (PTGS) phenomena, knocking down specific gene expression with high potency and less toxicity than traditional antisense gene therapies.
- PTGS post-transcriptional gene silencing
- dsRNA interferon-induced global RNA degradation when the dsRNA size is larger than 25 base pairs (bp), especially in mammalian cells.
- siRNA short interfering RNA
- miRNA microRNA
- the size limitation impairs the usefulness of RNAi, as it is difficult to deliver such small and unstable dsRNAs in vivo due to high dsRNase activities in human bodies (Brantl (2002) Biochimica et Biophysica Acta 1575: 15-25). Therefore, there remains a need for a more effective and reliable gene modulation system.
- This invention is based, at least in part, on the discovery that an artificial intron can be used to regulate the function of a gene in a cell.
- the invention features an isolated RNA comprising an intron RNA.
- the intron RNA is released in a cell (e.g., a mammalian cell), thereby modulating the function of a target gene.
- the isolated RNA does not contain a combination of a splice donor site that includes 5′-GU(A/G)AGU-3′ and a splice acceptor site that includes 5′-CU(A/G)A(C/U)NG-3′ (N is A, G, C, or U).
- a splice donor site that includes 5′-GUA(A/-)GAG(G/U)-3′ (“-” designates an empty position
- a splice acceptor site
- the splice donor site can be 5′-AGGUAAGAGGAU-3′ (SEQ ID NO:4), 5′-AGGUAAGAGU-3′ (SEQ ID NO:5), 5′-AGGUAGAGU-3′, or 5′-AGGUAAGU-3′;
- the splice acceptor site can be 5′-GAUAUCCUGCAGG-3′ (SEQ ID NO:6), 5′-GGCUGCAGG-3′, or 5′-CCACAGC-3′;
- the branch site can be 5′-UACUAAC-3′ or 5′-UACUUAUC-3′.
- the isolated RNA can be introduced into a cell for control of a gene function.
- the invention also provides a DNA template for the isolated RNA of the invention, an expression vector comprising the DNA, a cultivated cell comprising the isolated RNA or the DNA, an animal (e.g., a mammal such as a mouse) comprising the isolated RNA or the DNA, and a composition comprising the isolated RNA or the DNA.
- the invention further provides a method of producing an intron RNA.
- the method comprises cultivating the above-described cell to allow expression and/or release of the intron RNA.
- the released intron RNA can be left in the cell for control of a gene function, or be collected from the cell and used for generation of a DNA-RNA hybrid or delivery into another cell.
- Also within the scope of the invention is a method of modulating the function of a target gene in a cell.
- the method comprises introducing into a cell an effective amount of the isolated RNA or DNA of the invention.
- the intron RNA is then released in the cell, thereby modulating the function of a target gene.
- the invention features a composition
- a composition comprising a chemokine (e.g., interleukin-2) and an isolated RNA or a DNA as described above.
- a chemokine e.g., interleukin-2
- an isolated RNA or a DNA as described above.
- an effective amount of this composition can be administering into a cell (e.g., a mammalian cell or a cell infected by a virus) to modulate the function of a target gene.
- a cell e.g., a mammalian cell or a cell infected by a virus
- an HIV-1-infected cell can be treated with a combination of interleukin-2 and an isolated RNA containing an intron RNA complementary to an HIV-1 genomic sequence.
- the intron RNA induces degradation of the HIV-1 genomic sequence or its derivatives, or prevent it from being translated into polypeptides.
- the invention features a composition comprising one or more agents that induce RNA-mediated modulation of the functions of two or more target genes in a cell (e.g., a mammalian cell or a cell infected by a virus).
- a method of modulating the functions of genes in a cell by administering into the cell an effective amount of the composition is also within the scope of the invention.
- a cell when a cell is infected by HIV-1, it can be treated with one or more DNA-RNA hybrids or exogenous intron RNAs that cause degradation of HIV-1 RNAs, cellular RNAs such as Naf1 ⁇ , Nb2HP, and Tax1BP RNAs, or their derivatives, or prevent these RNAs from being translated into polypeptides.
- the present invention provides compositions and methods for treating human diseases. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and the accompanying drawings, and from the claims.
- FIG. 1 depicts a novel strategy for producing desired RNA molecules in cells through RNA splicing.
- FIG. 2 depicts generation of antisense RNAs by spliceosome cleavage of retroviral (e.g., LTR) promoter-mediated precursor transcripts.
- retroviral e.g., LTR
- FIG. 3 depicts generation of sense and antisense siRNAs by spliceosome cleavage of viral (e.g., CMV or AMV) promoter-mediated precursor transcripts.
- viral e.g., CMV or AMV
- FIG. 4 depicts generation of hairpin RNAs by spliceosome cleavage of Pol II (e.g., TRE or Tet response element) promoter-mediated precursor transcripts.
- Pol II e.g., TRE or Tet response element
- FIG. 5 depicts microscopic results, showing interference with green fluorescent protein (eGFP) expression in rat neuronal stem cells by various SpRNAi constructs.
- eGFP green fluorescent protein
- FIG. 6 depicts Western blot results, showing interference with green fluorescent protein (eGFP) expression in rat neuronal stem cells by various SpRNAi constructs.
- eGFP green fluorescent protein
- FIG. 7 depicts Western blot results, showing interference with integrin ⁇ 1 (ITGb1) expression in human prostatic cancer LNCaP cells by various SpRNAi constructs.
- FIG. 9 depicts potential differences between traditional PTGS/RNAi and SpRNAi phenomena.
- FIGS. 10A-B depict experimental evidence for generation of D-RNAi-induced miRNA.
- FIGS. 11A-C depict in vivo gene silencing by anti- ⁇ -catenin D-RNAi in embryonic chicken.
- FIG. 12 depicts in vivo gene silencing by anti-tyr D-RNAi in mouse.
- FIG. 1 shows an example of a novel strategy for producing a desired RNA molecule in a cell after RNA splicing event occurs.
- the desired RNA molecule like a natural intron, is flanked by an RNA splicing donor and an acceptor site.
- the DNA template for the desired RNA is inserted into a gene, which is expressed by type-II RNA polymerase (Pol II) transcription machinery under the control of either Pol II or viral RNA promoter.
- Pol II type-II RNA polymerase
- the desired RNA molecule is an antisense RNA that serves as an antisense oligonucleotide probe for antisense gene therapy.
- the desired RNA molecule is translated into a polypeptide that is useful in gene replacement therapy.
- the desired RNA molecule can also consist of small antisense and sense RNA fragments to function as double-stranded siRNA for RNAi induction.
- the desired RNA molecule can be a small hairpin-like RNA capable of causing RNAi-associated gene silencing phenomena.
- the desired RNA molecule can also be a ribozyme. All of the above desired RNA molecules are produced by intracellular splicing events and therefore named as “SpRNAi” for convenience.
- the invention features an isolated RNA comprising an intron RNA that is released in a cell, thereby modulating the function of a target gene.
- An “isolated RNA” is a ribonucleic acid the structure of which is not identical to that of any naturally occurring ribonucleic acid or to that of any fragment of a naturally occurring ribonucleic acid.
- a “function of a target gene” refers to the capability of the target gene to be transcribed into an RNA, the capability of the RNA to be stabilized, processed (e.g., through splicing), reverse transcribed or translated, and the capability of the RNA to play its normal role, e.g., serving as a tRNA and rRNA.
- RNA splicing is a process that removes introns and joins exons in a primary transcript.
- the structures of intron RNAs are well known in the art.
- An intron usually contains signal sequences for splicing. For example, most introns start from the sequence GU and end with the sequence AG (in the 5′ to 3′ direction), which are referred to as the splice donor and splice acceptor site, respectively.
- an intron has a branch site between the donor and the acceptor site. The branch site contains an A residue (branch point), which is conserved in all genes.
- the exon sequence is (A/C)AG at the 5′-exon-intron junction, and is G at the 3′-exon-intron junction.
- the fourth element is a poly-pyrimidine tract located between the branch site and the acceptor site.
- the splice donor site may contain 5′-GUA(A/-)GAG(G/U)-3′
- the splice acceptor site may contain 5′-G(A/U/-)(U/G)(C/G)C(U/C)(G/A)CAG-3′
- a branch site may contain 5′-UACU(A/U)A(C/U)(-/C)-3′
- a poly-pyrimidine tract may contain 5′-(U(C/U)) 1-3 (C/-)U 7-12 C(C/-)-3′ or 5′-(UC) 7-12 NCUAG(G/-)-3′.
- the intron RNA serves as or is farther processed to become, e.g., an RNA encoding a polypeptide, or an antisense RNA, short-temporary RNA (stRNA), microRNA (miRNA), small-interfering RNA (siRNA), short-hairpin RNA (shRNA), long deoxyribonucleotide-containing RNA (D-RNA), or ribozyme RNA, each of which may be in either sense or antisense orientation.
- stRNA short-temporary RNA
- miRNA microRNA
- siRNA small-interfering RNA
- shRNA short-hairpin RNA
- D-RNA long deoxyribonucleotide-containing RNA
- ribozyme RNA each of which may be in either sense or antisense orientation.
- the intron RNA region homologous or complementary to its target gene ranges from 14 to 2,000 nucleotides, most preferably between 19 and 500 nucleotides.
- the intron RNA may be 35-100% (i.e., any integral between and including 35 and 100) identical or complementary to its target gene.
- the preferred homology or complementarity is 35-65% and more preferably 41-49% for an shRNA, 40-100% and more preferably 90-100% for a sense or antisense RNA.
- the length of an siRNA/miRNA/shRNA may be 16-38 nucleotides, and preferably 19-25 nucleotides. Additionally, there may be one or more linker sequences, e.g., between the donor and the acceptor site and the antisense RNA, stRNA, siRNA, shRNA, D-RNA or ribozyme RNA sequence.
- the isolated RNA may further contain exons encoding a polypeptide for co-expression with the intron RNA.
- the polypeptide may be a normal protein, a missing protein, a dominant-negative protein, or a protein marker such as a fluorescent protein, luciferase, or lac-Z.
- RNA of the invention can be chemically synthesized or produced by transcription from a DNA template in vitro and in vivo.
- the template DNA can be cloned into an expression vector according the methods well known in the art.
- vectors include, but are not limited to, plasmids, cosmids, phagemids, yeast artificial chromosome, retroviral vectors, lentiviral vectors, lambda vector, adenoviral (AMV) vector, adeno-associated viral (AAV) vector, hepatitis virus (HBV)-modified vector, and cytomegalovirus (CMV)-related viral vector.
- the isolated RNA, DNA template, and expression vector described above can be introduced into a cultured cell or a subject (e.g., an animal or a human) using methods commonly employed in the art such as transfection, infection, electroporation, micro-injection, and gene-gun penetration.
- the isolated RNA, DNA template, and expression vector may be formulated into a composition.
- the intron RNA, once expressed and/or released in the cell, can modulate the function of a target gene, for example, inhibit a cancer-related gene, potential viral gene, and microbe-related gene. Therefore, this method is useful for treating and preventing diseases such as cancer and viral or microbial infection.
- a composition is suspended in a pharmaceutically acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
- a pharmaceutically acceptable carrier e.g., physiological saline
- the dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the subject's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100.0 ⁇ g/kg. Wide variations in the needed dosage are to be expected in view of the variety of compounds available and the different efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
- a suitable delivery vehicle e.g., polymeric microparticles or implantable devices
- composition may be delivered to the subject, for example, by use of polymeric, biodegradable microparticle or microcapsule delivery devices known in the art.
- liposomes prepared according to standard methods.
- the vectors can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies.
- Poly-L-lysine binds to a ligand that can bind to a receptor on target cells (Cristiano et al. (1995) J. Mol. Med. 73: 479).
- tissue specific targeting can be achieved by use of tissue-specific transcriptional regulatory elements (TRE) which are known in the art.
- Delivery of naked nucleic acids (i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site is another means to achieve in vivo expression.
- an “effective amount” is an amount of the compound or composition that is capable of producing a medically desirable result (e.g., a decreased expression level of a cancer-related gene, potential viral gene, or microbe-related gene) in a treated subject.
- an animal comprising an isolated RNA or a DNA of the invention can be produced according to the methods described above or any other methods known in the art.
- a “knock-out animal” may be generated in which a target gene is partially (e.g., only in some tissues) or completely inhibited.
- the animal can be a farm animal such as a pig, goat, sheep, cow, horse and rabbit, a rodent such as a rat, guinea pig, and mouse, or a non-human primate such as a baboon, monkey, and chimpanzee.
- These animals of the invention can be used as disease models.
- these animals can be used to identify a compound or composition effective for treatment or prevention of a disease.
- Compounds or compositions can be identified by administering a test compound or composition to a model animal or by contacting the test compound or composition with an organ, a tissue or cells derived from a model animal. Effects of the test compound or composition on the disease of the animal, organ, tissue or cells are evaluated. Test compounds or compositions that palliate the disease symptoms can be effective for treatment or prevention of the disease.
- a second aspect of the invention is based on the discovery that the combination of interleukin-2 and a viral RNA-antisense DNA hybrid significantly reduced human immunodeficiency virus-1 (HIV-1) subtype B gene activity. Consequently, the invention features a composition comprising a chemokine and an isolated RNA or a DNA of the invention.
- the isolated RNA or DNA allows an intron RNA to be released in a cell, thereby modulating the function of a target gene.
- chemokines include, but are not limited to, interleukin-2 (IL-2), interleukin-10 (IL-10), interleukin-17 (IL-17), tumor narcosis factor- ⁇ (TNF- ⁇ ), and tumor narcosis factor- ⁇ (TNF- ⁇ ).
- the intron RNA may contain, e.g., an antisense RNA, stRNA, miRNA, siRNA, shRNA, D-RNA, or ribozyme RNA.
- the composition can be administering into a cell according to the methods described above for modulating the function of a target gene in a cell, e.g., inducing degradation of an HIV-1 genomic sequence or preventing an HIV-1 genomic sequence from being translated into a polypeptide in an HIV-1 infected cell.
- the invention therefore provides a composition comprising one or more agents (e.g., an antisense RNA, stRNA, miRNA, siRNA, shRNA, D-RNA, SpRNAi, ribozyme RNA, or a combination thereof) that induce RNA-mediated modulation of the functions of two or more target genes in a cell.
- agents e.g., an antisense RNA, stRNA, miRNA, siRNA, shRNA, D-RNA, SpRNAi, ribozyme RNA, or a combination thereof
- the composition can be administering into a cell according to the methods described above for control of the functions of genes.
- Applications of the present invention include, without limitation, therapy by suppression of cancer-related genes, vaccination against potential viral genes, treatment of microbe-related genes, research of candidate molecular pathways with systematic knockout/knockdown of involved molecules, and high-throughput screening of gene functions based on microarray analysis.
- the present invention can also be used as a tool for studying gene function under physiological and therapeutical conditions, providing compositions and methods for altering the characteristics of eukaryotic cells such as cancerous, virus-infected, microbe-infected, physiologically diseased, genetically mutated, and pathogenic cells.
- Rat neuronal stem cell clones AP31 and PZ5a were primary-cultured and maintained as described by Palmer et al. (1999) J. Neuroscience 19: 8487-8497.
- the cells were grown on polyornathine/laminin-coated dishes in DMEM/F-12 (1:1; high glucose) medium containing 1 mM ⁇ L-glutamine supplemented with 1 ⁇ N2 supplements (Gibco/BRL, Gaithersburg, Md.) and 20 ng/ml FGF-2 (Invitrogen, Carlsbad, Calif.) without serum at 37° C. under 5% CO 2 .
- 75% of the medium was replaced with new growth medium every 48 h.
- the LNCaP culture was passaged at ⁇ 80% confluency by exposing cells to trypsin-EDTA solution for 1 min and rinsing them once with RPMI, and detached cells were replated at 1:10 dilution in fresh growth medium. After a 48-hour incubation period, RNA was isolated from the cells using RNeasy spin columns (Qiagen, Valencia, Calif.), fractionated on a 1% formaldehyde-agarose gel, and transferred onto nylon membranes. The genomic DNA was also isolated using apoptotic DNA ladder kit (Roche Biochemicals, Indianapolis, Ind.) and assessed by 2% agarose gel electrophoresis, while cell growth and morphology were examined using microscopy and cell counting.
- Synthetic nucleic acid sequences used for generation of three different SpRNAi introns containing either sense, antisense or hairpin eGFP insert were as follows: N1-sense, 5′-pGTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA GA-3′ (SEQ ID NO:7); N1-antisense, 5′-pCGCGTCTTGA AGAAGATGGT GCGCTCCTGC GATCGGATCC TCTTAC-3′ (SEQ ID NO:8); N2-sense, 5′-pGTAAGAGGAT CCGATCGCTT GAAGAAGATG GTGCGCTCCT GA-3′ (SEQ ID NO:9); N2-antisense, 5′-pCGCGTCAGGA GCGCACCATC TTCTTCAAGC GATCGGATCC TCTTAC-3′ (SEQ ID NO:10); N3-sense, 5′-pGTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA GTTAACTTGA AG
- rGFP refers to a new red-fluorescin chromoprotein generated by insertion of an additional aspartate at amino acid (aa) 69 of HcRed1 chromoprotein from Heteractis crispa . (Gurskaya et al. (2001) FEBS Letters 507: 16-20), developing less aggregate and almost twice intense far-red fluorescent emission at ⁇ 570-nm wavelength.
- This mutated rGFP gene sequence was cloned into pHcRed1-N1/1 plasmid vector (BD Biosciences) and propagated in E. coli DH5 ⁇ in LB medium containing 50 ⁇ g/ml kanamycin (Sigma).
- the pHcRed1-N1/1 plasmid was cleaved with XhoI and XbaI restriction enzymes.
- a 769-bp rGFP fragment and a 3,934-bp empty plasmid were purified separately from a 2% agarose gel after electrophoresis.
- Hybridization of N1-sense to N1-antisense, N2-sense to N2-antisense, N3-sense to N3-antisense, and N4-sense to N4-antisense was separately performed by heating each mixture of complementary sense and antisense (1:1) sequences to 94° C. for 2 min and then 70° C. for 10 min in 1 ⁇ PCR buffer (e.g., 50 mM Tris-HCl, pH 9.2 at 25° C., 16 mM (NH 4 ) 2 SO 4 , 1.75 mM MgCl 2 ).
- 1 ⁇ PCR buffer e.g., 50 mM Tris-HCl, pH 9.2 at 25° C., 16 mM (NH 4 ) 2 SO 4 , 1.75 mM MgCl 2 ).
- ligation of the N1, N2 or N3 hybrid to the N4 hybrid was performed by gradually cooling a mixture of N1-N4, N2-N4 or N3-N4 (1:1) hybrids from 50° C. to 10° C. over a period of 1 h, and then T 4 ligase and buffer (Roche) were mixed with the mixture for 12 h at 12° C. so as to obtain introns for linking to exons with proper ends. After the rGFP exon fragments were added into the reaction (1:1:1), T4 ligase and buffer were adjusted accordingly for continued ligation for another 12 h at 12° C.
- rGFP-specific primers 5′-dCTCGAGCATG GTGAGCGGCC TGCTGAA-3′ (SEQ ID NO:15) and 5′-dTCTAGAAGTT GGCCTTCTCG GGCAGGT-3′ (SEQ ID NO:16) at 94° C., 1 min; 52° C., 1 min; and then 68° C., 2 min for 30 cycles.
- the PCR products were fractionated on a 2% agarose gel, and a ⁇ 900-bp nucleotide sequence was extracted and purified using a gel extraction kit (Qiagen). The composition of this ⁇ 900 bp SpRNAi-eGFP-containing rGFP gene was confirmed by sequencing.
- the recombinant gene Since the recombinant gene possesses an XhoI and an XbaI restriction site at its 5′- and 3′-end, respectively, it can be easily cloned into a vector with ends complementary to the XhoI and XbaI sites.
- the vector can be an expression vector, e.g., a plasmid, cosmid, phagmid, yeast artificial chromosome, or viral vector.
- the insert within the intron is flanked by a PvuI and an MluI restriction site at its 5′- and 3′-end, respectively, the insert can be removed and replaced with another insert with ends complementary to the PvuI and MluI sites.
- the insert sequence can be homologous or complementary to a gene fragment such as a fluorescent protein gene, luciferase gene, lac-Z gene, plant gene, viral genome, bacterial gene, animal gene, and human oncogene.
- the homology and/or complementarity ranges from about 30 ⁇ 400%, more preferably 35 ⁇ 49% for a hairpin-shRNA insert and 90 ⁇ 100% for both sense-siRNA and antisense-siRNA inserts.
- the SpRNAi-recombinant rGFP gene and the linearized 3,934-bp empty pHcRed1-N1/1 plasmid were mixed at 1:16 (w/w) ratio.
- the mixture was cooled from 65° C. to 15° C. over a period of 50 min, and then T 4 ligase and buffer were added into the mixture for ligation at 12° C. for 12 h.
- a so formed SpRNAi-recombinant rGFP-expressing plasmid vector was propagated in E. coli DH5 ⁇ in LB medium containing 50 ⁇ g/ml kanamycin.
- a positive clone was confirmed by PCR with rGFP-specific primers SEQ ID NO:15 and SEQ ID NO:16 at 94° C., 1 min and then 68° C., 2 min for 30 cycles and subsequent sequencing.
- rGFP-specific primers SEQ ID NO:15 and SEQ ID NO:16 at 94° C., 1 min and then 68° C., 2 min for 30 cycles and subsequent sequencing.
- cloning into viral vectors the same ligation procedure was performed except that an XhoI/XbaI-linearized pLNCX2 retroviral vector (BD Biosciences) was used.
- the eGFP insert within the SpRNAi intron was removed and replaced with various integrin ⁇ 1-specific insert sequences with ends complementary to the PvuI and MluI sites.
- Synthetic nucleic acid sequences used for generation of various SpRNAi introns containing either sense, antisense or hairpin integrin ⁇ 1 insert were as follows P1-sense, 5′-pCGCAAGCAGG GCCAAATTGT GGGTA-3′ (SEQ ID NO:17); P1-antisense, 5′-pTAGCACCCAC AATTTGGCCC TGCTTGTGCG C-3′ (SEQ ID NO:18); P2-sense, 5′-pCGACCCACAA TTTGGCCCTG CTTGA-3′ (SEQ ID NO:19); P2-antisense, 5′-pTAGCCAAGCA GGGCCAAATT GTGGGTTGCG C-3′ (SEQ ID NO:20); P3-sense, 5′-pCGCAAGCAGG GCCAAATTGT GGGTTTAAAC CCACAATTTG GCCCTGCTTG A-3′ (SEQ ID NO:21); P3-antisense, 5′-pTAGCACCCAC AATTTGGCCC TGCTTGAATT
- inserts were designed using Gene Runner software v3.0 (Hastings, Calif.) and formed by hybridization of P1-sense to P1-antisense, P2-sense to P2-antisense and P3-sense to P3-antisense for targeting nt 244 ⁇ 265 of the integrin ⁇ 1 gene (GenBank Access No. NM 002211.2).
- the SpRNAi-containing rGFP-expressing retroviral vector was propagated in E. coli DH5 ⁇ in LB medium containing 100 ⁇ g/m ampcillin (Sigma).
- a packaging cell line PT67 (BD Biosciences) was also used for producing infectious, replication-incompetent viruses.
- Transfected PT67 cells were grown in DMEM medium supplemented with 10% fetal bovine serum with 4 mM L-glutamine, 1 mM sodium pyruvate, 100 ⁇ g/ml streptomycin sulfate and 50 mg/ml neomycin (Sigma) at 37° C. under 5% CO 2 .
- the titer of the virus produced by PT67 cells was determined to be at least 10 6 cfu/ml before transfection.
- RNA (20 ⁇ g total RNA or 2 ⁇ g poly[A + ] RNA) was fractionated on 1% formaldehyde-agarose gels and transferred onto nylon membranes (Schleicher & Schuell, Keene, N. H.).
- a synthetic 75-bp probe (5′-dCCTGGCCCCC TGCTGCGAGT ACGGCAGCAG GACGTAAGAG GATCCGATCG CAGGAGCGCA CCATCTTCTT CAAGT-3′ (SEQ ID NO:23) targeting the junction region between rGFP and the hairpin eGFP-insert was labeled with the Prime-It II kit (Stratagene, La Jolla, Calif.) by random primer extension in the presence of [ 32 P]-dATP (>3000 Ci/mM, Amersham International, Arlington Heights, Ill.), and purified using 30 bp-cutoff Micro Bio-Spin chromatography columns (Bio-Rad, Hercules, Calif.).
- Hybridization was carried out in a mixture of 50% freshly deionized formamide (pH 7.0), 5 ⁇ Denhardt's solution, 0.5% SDS, 4 ⁇ SSPE and 250 mg/mL denatured salmon sperm DNAs (18 h, 42° C.). Membranes were sequentially washed twice in 2 ⁇ SSC, 0.1% SDS (15 min, 25° C.), and once in 0.2 ⁇ SSC, 0.1% SDS (15 min, 25° C.) before autoradiography.
- rat neuronal stem cells were transfected with SpRNAi-recombinant rGFP plasmids encoding either a sense, antisense or hairpin eGFP insert using Fugene reagent (Roche). Plasmids containing insert-free rGFP gene and SpRNAi-recombinant rGFP gene with an insert against HIV-gag p24 were used as negative controls. Cell morphology and fluorescent images were photographed at 0-, 24- and 48-hour time points after transfection. At the 48-h incubation time point, the rGFP-positive cells were sorted by flow cytometry and collected for Western blot analysis.
- LNCaP cells were transfected with pLNCX2 retroviral vectors containing various SpRNAi-recombinant rGFP genes against nt 244 ⁇ 265 of integrin ⁇ 1 using the Fugene reagent.
- the transfection rate of pLNCX2 retroviral vector into LNCaP cells was determined to be about 20%, while the pLNCX2 virus was less infectious to LNCaP cells. The same analyses were performed as aforementioned.
- Protein fractions were electroblotted onto a nitrocellulose membrane, blocked with Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) for 1 ⁇ 2 h at room temperature.
- GFP expression was assessed using primary antibodies directed against eGFP (1:5,000; JL-8, BD Biosciences, Palo Alto, Calif.) or rGFP (1:10,000; BD Biosciences) overnight at 4° C.
- the blot was then rinsed 3 times with TBS-T and exposed to a secondary antibody, goat anti-mouse IgG conjugate with Alexa Fluor 680 reactive dye (1:2,000; Molecular Probes), for 1 h at room temperature.
- RNA-polymerase cycling reaction RNA-PCR
- RNA-PCR RNA-polymerase cycling reaction
- an SpRNAi-recombinant gene source an SpRNAi-sense HIV recombinant gene containing a sequence homologues to HIV-1 genome from +2113 to +2453 bases was generated following a procedure similar to Section 2 above.
- RNA product (10 ⁇ 50 ⁇ g) of the SpRNAi-sense HIV recombinant gene were transcribed in about 10 6 transfected cells, isolated using RNeasy columns (Qiagen), and then hybridized to its pre-synthesized complementary DNA (cDNA) by heating and then cooling the mixture from 65° C. to 15° C. over a period of 50 min. Transfection was performed following the same procedure shown in Section 5 above.
- RNA splicing/processing-directed gene silencing was tested using an artificial recombinant gene, SpRNAi-rGFP ( FIG. 1 ).
- SpRNAi splicing-competent intron
- rGFP red fluorescin gene
- pre-rRNA pre-ribosomal RNA
- AS acceptor
- PPT poly-pyrimidine tract
- DNA templates for splicing-competent introns were synthesized and inserted into an intron-free red fluorescin gene (rGFP) that was mutated from the HcRed1 chromoprotein of Heteractis crispa . Since the inserted intron disrupted the functional fluorescin structure of the rGFP protein, occurrence of intron splicing and rGFP-mRNA maturation was indicated by the reappearance of red fluorescent light emission at the 570-nm wavelength in a transfected cell.
- rGFP intron-free red fluorescin gene
- SpRNAi was based on the natural structure of a pre-messenger RNA intron, consisting of spliceosome-dependent nucleotide components, such as donor and acceptor splicing sites in both ends for precise cleavage, branch point domain for splicing recognition, poly-pyrimidine tract for spliceosome interaction, linkers for connection of each major components and some artificially added multiple restriction/cloning sites for cloning of inserts.
- spliceosome-dependent nucleotide components such as donor and acceptor splicing sites in both ends for precise cleavage, branch point domain for splicing recognition, poly-pyrimidine tract for spliceosome interaction, linkers for connection of each major components and some artificially added multiple restriction/cloning sites for cloning of inserts.
- the donor splicing site is an oligonucleotide sequence either containing or homologous to 5′-exon-AG-(splicing point)-GTA(A/-)GAG(G/T)-3′ (SEQ ID NO:24), e.g., 5′-AG GTAAGAGGAT-3′ (SEQ ID NO:25), 5′-AG GTAAGAGT-3′ (SEQ ID NO:26), 5′-AG GTAGAGT-3′, 5′-AG GTAAGT-3′ and so on.
- the acceptor splicing site is an oligonucleotide sequence either containing or homologous to 5′-G(W/-)(T/G)(C/G)C(T/C)(G/A)CAG-(splicing point)-G/C-exon-3′ (while W is a pyrimidine, i.e., A or T) (SEQ ID NO:27), e.g., 5′-GATATCCTGCAG G-3′ (SEQ ID NO:28), 5′-GGCTGCAG G-3′, 5′-CCACAG C-3′ and so on.
- the branch point is an “A” residue located within a sequence homologous to 5′-TACT(A/T)A*(C/T)(-/C)-3′ (while the symbol “*” marks the branch site), e.g., 5′-TACTAAC-3′, 5′-TACTTATC-3′ and so on.
- the poly-pyrimidine tract is a high T and/or C content oligonucleotide sequence homologous to 5′-(TY)m(C/-)(T)nC(C/-)-3′ or 5′-(TC)nNCTAG(G/-)-3′ (while Y is a C or T).
- T deoxythymidine
- U uridine
- RNAs DNA templates for aberrant RNAs, e.g., short-temporary RNA (stRNA), small-interfering RNA (siRNA), short-hairpin RNA (shRNA), long deoxyribonucleotide-containing RNA (D-RNA) and potentially ribozyme RNA in either sense or antisense orientation.
- stRNA short-temporary RNA
- siRNA small-interfering RNA
- shRNA short-hairpin RNA
- D-RNA long deoxyribonucleotide-containing RNA
- ribozyme RNA e.g., ribozyme RNA in either sense or antisense orientation.
- the gene silencing effect of a hairpin-RNA-containing SpRNAi is stronger than that of a sense- and antisense-RNA-containing SpRNAi, showing an average of >80% knockdown specificity to all targeted gene products.
- knockdown specificity is mainly determined by the homologous or complementary region of an insert to the
- the tested hairpin-SpRNAi insert had about 40 ⁇ 42% homology and another 40 ⁇ 42% complementarity to the targeted gene domain, with-in-between of which an A/T-rich linker sequence filled in the rest 8 ⁇ 10% space.
- the homology or complementarity can be increased up to 100%, an average of 40 ⁇ 50% knockdown efficacy was detected in most of the transfection tests.
- different types of SpRNAi inserts and/or the combination thereof can be used to manipulate specific gene expression levels in cells.
- SpRNAi-containing genes were cloned into an expression-competent vector, e.g., plasmid, cosmid, phagmid, yeast artificial chromosome, viral vector and so on. As shown in FIGS.
- the vectors can contain at least one viral or type-II RNA polymerase (Pol II) promoter or both for the expressing of the SpRNAi-gene in eukaryotic cells, a Kozak consensus translation initiation site to increase translation efficiency in eukaryotic cells, SV40 polyadenylation signals downstream of the SpRNAi-gene for processing of the 3′-end gene transcript, a pUC origin of replication for propagation in prokaryotic cells, at least two multiple restriction/cloning sites for cloning of the SpRNAi-gene, an optional SV40 origin for replication in mammalian cells expressing the SV40 T antigen and an optional SV40 early promoter for expressing an antibiotic resistance gene in replication-competent prokaryotic cells.
- a viral or type-II RNA polymerase (Pol II) promoter or both for the expressing of the SpRNAi-gene in eukaryotic cells a Kozak consensus translation initiation site to increase translation efficiency in eukaryotic cells
- antibiotic resistance genes was used as a selective marker for searching of successfully transfected or infected clones that are resistance to the antibiotics such as penicillin G, ampcillin, neomycin, paromycin, kanamycin, streptomycin, erythromycin, spectromycin, phophomycin, tetracycline, rifapicin, amphotericin B, gentamicin, chloramphenicol, cephalothin, tylosin and the combination thereof.
- antibiotics such as penicillin G, ampcillin, neomycin, paromycin, kanamycin, streptomycin, erythromycin, spectromycin, phophomycin, tetracycline, rifapicin, amphotericin B, gentamicin, chloramphenicol, cephalothin, tylosin and the combination thereof.
- the vector was therefore stable enough to be introduced into a cell(s), tissue or animal body using a highly efficient gene delivery method, e.g., liposomal transfection, chemical transfection, chemical transformation, electroporation, infection, micro-injection and gene-gun penetration.
- a highly efficient gene delivery method e.g., liposomal transfection, chemical transfection, chemical transformation, electroporation, infection, micro-injection and gene-gun penetration.
- knockdown levels of eGFP protein in rat neuronal stem cell clones AP31 and PZ5a by various SpRNAi inserts were measured on an unreduced 6% SDS-polyacrylamide gel.
- rGFP protein levels ⁇ 30 kDa, red bars
- Y axis an average expression range of 82 ⁇ 100% intensity
- eGFP levels (27 kDa, green bars) were found to be reduced by transfection of SpRNAi-rGFP genes containing sense-eGFP (43.6%), antisense-eGFP (49.8%) or hairpin-eGFP (19.0%) inserts, but not that of intron-free rGFP gene (blank control) or SpRNAi-rGFP gene containing hairpin-HIV p24 insert (negative control).
- the ITGb1 levels (29 kDa) were significantly reduced by transfection of SpRNAi-rGFP genes containing a sense-ITGb1 (37.3%), antisense-ITGb1 (48.1%) and hairpin-ITGb1 (13.5%) inserts, but not that of an intron-free rGFP gene (blank control) or SpRNAi-rGFP gene containing a hairpin-HIV p24 insert (negative control).
- RNA-directed endoribonuclease activity was mildly increased up to 6.7% by the transfection, while no interferon-induced cytotoxicity was detected.
- the cellular genes corresponding to combinational therapy have been, further investigated by microarray analysis for AIDS prevention. Co-suppression of three microarray-identified target genes, Naf1 ⁇ , Nb2 protein homologous to Wnt-6 and Tax1 binding protein was shown to prevent an average of 80.2% HIV-1b entry and infection in a primary CD4 + T cell model.
- vRNA viral RNA
- aDNA hybrid construct was designed to silence an early-stage gene locus containing gag/pol/pro viral genes and p24 HIV-1 gene marker.
- the anti-gag/pol/pro transfection interferes with the integration of viral provirus into host chromosome and also prevents the activation of several viral genes, while the anti-p24 transfection provides a visual indicator for observing viral activity on an ELISA assay. The results showed that such strategy was effective in knocking out exogenous viral gene expression ex vivo in a CD4 + T lymphocyte extract model.
- PBMC Peripheral blood mononuclear cells extracted from patients were purified using CD4 + -affinity immunomagnetic beads and grown in RPMI 1640 medium with 200 U/ml IL-2 adjuvant treatment for more than two weeks.
- Pure HIV-1 provirus was shown as a viral genome sized about 9.7 kb on a formaldehyde-containing RNA electrophoresis gel.
- Samples of CD4 + Th lymphocyte RNA extracts from normal, non-infected persons were used as negative controls, while those from HIV-1 infected patients were used as positive controls. No significant gene silencing effect was detected in all controls or transfections of other constructs, including vDNA-aRNA hybrid of HIV-1b, aDNA only and vRNA-aDNA against HTLV-1 rather than HIV-1.
- FIG. 8 Northern blot analysis of SpRNAi-induced gene silencing effects on Naf1 ⁇ , Nb2HP and Tax1BP was shown to prevent HIV-1 type B infection.
- the tested gene targets were selected through RNA-PCR microarray analysis of differential expression genes from the acute (one ⁇ two weeks) and chronic (about two years) infected patients' primary T cells with or without 25 nM anti-HIV D-RNAi treatment (Lin et al. (2001) supra).
- the SpRNAi product concentrations in all treatments were normalized to 30 nM.
- FIG. 8B is a bar chart showing HIV-gag p24 ELISA results (white) in correlation with the treatment results demonstrated in FIG. 8A .
- CD4 may not be an ideal target for HIV prevention.
- the search for HIV-dependent cellular genes in vivo was hindered by the fact that infectivity of viruses and infection rate among different patients are usually different and lead to inconsistent results.
- Short-term ex vivo culture conditions can normalize infectivity and infection rate of HIV transmission in a more uniformed CD4 + T cell population.
- Microarray analysis based on such ex vivo conditions would be reliable for critical biomedical and genetic research of HIV-1 infection.
- Microarray studies identified differential gene profiles between HIV ⁇ and HIV + T cells in the acute and chronic infection phases and provided many potential anti-HIV cellular gene targets for AIDS therapy and prevention.
- the p58/HHR23 species that codes for XP-C repair-complementing proteins is a human homologue of yeast RAD23 derivatives, sharing an ubiquitin-like N-terminus. Based on its molecular similarity shared with RNA repairing-directed transcriptional regulation, the repair-complementing machinery indicates a novel mechanism of posttranscriptional gene silencing in addition to RNA interference.
- the newly recombined mRNA part of the D-RNAi agent may be further processed by intracellular Pol II and some unknown RNA excision machineries to generate miRNA-like molecules for long-term gene silencing.
- Pol II intracellular Pol II and some unknown RNA excision machineries to generate miRNA-like molecules for long-term gene silencing.
- both D-RNAi-derived small RNAs and Pol II RNA splicing-processed intron fragments have an average length of 15-45 nucleotides, which is comparable to the general sizes of Dicer-processed miRNA intermediates.
- both kinds of small RNAs isolated by guanidinium-chloride ultracentrifugation can elicit strong, but short-term gene silencing effects to genes homologous to the small RNAs in cells, indicating the possible miRNA-related interfering property of these small RNAs.
- the small miRNA-like RNAs are constitutionally derived from the large templates of mRNA-cDNA or precursor mRNA-genomic DNA hybrids, the long-term effect of D-RNAi phenomena may be maintained by accumulation of sufficient small miRNA-like RNAs rather than the stability of small RNAs. This also explains the delayed initiation phase observed in the D-RNAi-induced gene silencing and intron splicing-mediated PTGS phenomena (Lin et al. (2001) Biochem. Biophys. Res. Commun. 281: 639-644; and Lin et al. (2003) Biochem. Biophys. Res. Commun ., in press).
- an artificial intron mimicking the natural structure of a pre-mRNA intron was constructed for evaluating splicing-directed small RNA generation (Lin et al. (2003) Biochem. Biophys. Res. Commun ., in press).
- the splicing-competent artificial intron, SpRNAi is flanked by a splice donor (DS) and acceptor (AS) site, and contains a branch-point domain (BrP), a poly-pyrimidine tract (PPT) and at least one intronic insert located in the 5′-proximal domain of the artificial intron.
- DS splice donor
- AS acceptor
- BrP branch-point domain
- PPT poly-pyrimidine tract
- the SpRNAi also contains a translation stop codon in its 3′-proximal region, which if present in a cytoplasmic mRNA, would signal the diversion of the defective pre-mRNA from a non-sense mRNA degradation (NMD) pathway.
- NMD non-sense mRNA degradation
- the small miRNA-like RNAs are able to trigger translation repression or sometimes RNA degradation depending on the degree of complementarity and homology with their targets. According to the variety and complexity of natural miRNA structures, there is no artificial means to produce intracellular miRNA-like molecules before the finding of this intron splicing-mediated gene silencing phenomenon. The process of such miRNA-like small interfering RNA generation is therefore different from that for the dsRNA-induced RNAi; however, the possible involvement of RNAi mechanisms cannot be ruled out in that some small RNAs might form siRNAs by complementary hybridization within a localized compartment.
- D-RNAi can be used as an effective strategy to silence specific target gene in vivo.
- ⁇ -catenin gene was selected as an example because its product plays a critical role in the biological development and ontogenesis.
- ⁇ -catenin is known to be involved in the growth control of skin and liver tissues in chicken embryos.
- D-RNAi mRNA-cDNA hybrid
- the anti- ⁇ -catenin D-RNAi molecules were generated against the central region (aa 306-644) of the ⁇ -catenin coding sequence (GenBank Access No. X87838) by RNA-PCR.
- Fertilized eggs were obtained from SPAFAS farm (Preston, Conn.) and incubated in a humidified incubator. Using embryonic day 3 chicken embryos, a dose of 25 nM of the D-RNAi agent or reversal control hybrids of sense DNA-antisense RNA (sDNA-aRNA) was injected into the ventral body cavity, which is close to where the liver primordia would form ( FIG. 11A ). The mRNA-cDNA or the control sDNA-aRNA hybrids were mixed with DOTAP® liposomal transfection reagent (Roche) at a ratio of 3:2. A 10% (v/v) fast green solution was added before injection as a dye indicator.
- the mixtures were injected into the ventral side near the liver primordia and below the heart using heat pulled capillary needles. After injection, the eggs were sealed with scotch tape and put back into the humidified incubator at 39-40° C. until day 12 when the embryos were removed, examined and photographed under a dissection microscope. While there were malformations, the embryos survived and there was no visible overt toxicity or overall perturbation of embryo development.
- the liver was the closest organ to the injection site and was most dramatically affected in its phenotypes. Other regions, particularly the skin, were also affected by the diffused D-RNAi agent. As shown in FIG.
- the embryonic chicken livers After ten days of injection with the anti- ⁇ -catenin D-RNAi (mRNA-cDNA hybrid) agent, the embryonic chicken livers showed an enlarged and engorged first lobe, but the size of the second and third lobes of the livers were dramatically decreased ( FIG. 11C ). Histological sections of normal livers showed hepatic cords and sinusoidal space with few blood cells. In anti- ⁇ -catenin D-RNAi-treated embryos, the general architecture of the hepatic cells in lobes 2 and 3 remained unchanged; however, there were islands of abnormal regions in lobe 1. The endothelium development appeared to be defective and blood leaked outside of the blood vessels.
- anti- ⁇ -catenin D-RNAi mRNA-cDNA hybrid
- patched albino (white) skins of melanin-knockout mice were created by a succession of intra-cutaneous (i.c.) transduction of about 50 nM anti-tyrosinase (tyr) mRNA-cDNA hybrids for 4 days (a total of 200 nM).
- Tyr a type-I membrane protein and copper-containing enzyme, catalyzes the critical and rate-limiting step of tyrosine hydroxylation in the biosynthesis of melanin (black pigment) in skins and hairs.
- the D-RNAi transfections did not cause detectable cytotoxicity, while the dsRNA transfections induced noted non-specific mRNA degradation. This even underscores the fact that the mRNA-cDNA hybrids are effective even under in vivo systems without the side-effects of dsRNA. The results also indicate that this gene silencing effect is stable and efficient in knocking out target gene expression over a relatively long period of time since the hair regrowth requires at least a ten-day recovery. Further, it was observed that non-targeted skin hairs appeared to be normal, indicating that the compositions used herein possess high specificity and no overt toxicity. Thus, the D-RNAi-based gene manipulation offers the advantages of low in vivo dosage, stability, long-term effectiveness, and lack of overt toxicity.
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
An isolated RNA comprising an intron RNA that is released in a cell, thereby modulating the function of a target gene. Also disclosed are a composition comprising a chemokine and an isolated RNA of the invention or a DNA template for the isolated RNA, a composition comprising one or more agents that induce RNA-mediated modulation of the functions of two or more target genes in a cell, and methods of using these compositions for modulating the functions of genes in a cell.
Description
- This application claims priority to U.S. Provisional Application Ser. No. 60/411,062, filed Sep. 16, 2002, and U.S. Provisional Application Ser. No. 60/418,405, filed Oct. 12, 2002, the contents of which are incorporated herein by reference.
- This invention was made with support in part by a grant from NIH (CA 85722). Therefore, the U.S. government has certain rights.
- This invention relates to regulation of a gene function.
- One strategy for treating human diseases is to target specific disease-associated genes by either replacing impaired gene functions or by suppressing unwanted gene functions. Expression vectors are commonly used for introducing active genes into a cell to provide missing gene functions. To suppress unwanted gene functions, antisense oligonucleotides, antibodies, and small molecule drugs are often used as therapeutic agents.
- Applications of RNA interference (RNAi) (Elbashir et al. (2001) Nature 411: 494-498) and deoxyribonucleotidylated-RNA interfering (D-RNAi) (Lin et al. (2001) Biochem. Biophys. Res. Commun. 281: 639-644) technologies in treating human diseases are also in progress. RNAi elicits post-transcriptional gene silencing (PTGS) phenomena, knocking down specific gene expression with high potency and less toxicity than traditional antisense gene therapies. However, the gene silencing effects mediated by dsRNA are repressed by interferon-induced global RNA degradation when the dsRNA size is larger than 25 base pairs (bp), especially in mammalian cells. Although transfection of short interfering RNA (siRNA) or microRNA (miRNA) of less than 21 bp can overcome interferon-associated problems, the size limitation impairs the usefulness of RNAi, as it is difficult to deliver such small and unstable dsRNAs in vivo due to high dsRNase activities in human bodies (Brantl (2002) Biochimica et Biophysica Acta 1575: 15-25). Therefore, there remains a need for a more effective and reliable gene modulation system.
- This invention is based, at least in part, on the discovery that an artificial intron can be used to regulate the function of a gene in a cell.
- In one aspect, the invention features an isolated RNA comprising an intron RNA. The intron RNA is released in a cell (e.g., a mammalian cell), thereby modulating the function of a target gene. The isolated RNA does not contain a combination of a splice donor site that includes 5′-GU(A/G)AGU-3′ and a splice acceptor site that includes 5′-CU(A/G)A(C/U)NG-3′ (N is A, G, C, or U). It may contain a splice donor site that includes 5′-GUA(A/-)GAG(G/U)-3′ (“-” designates an empty position), a splice acceptor site that includes 5′-G(A/U/-)(U/G)(C/G)C(U/C)(G/A)CAG-3′ (SEQ ID NO:1), a branch site that includes 5′-UACU(A/U)A(C/U)(-/C)-3′, a poly-pyrimidine tract that includes 5′-(U(C/U))1-3(C/-)U7-12C(C/-)-3′ (SEQ ID NO:2) or 5′-(UC)7-12NCUAG(G/-)-3′ (SEQ ID NO:3), or a combination thereof. For example, the splice donor site can be 5′-AGGUAAGAGGAU-3′ (SEQ ID NO:4), 5′-AGGUAAGAGU-3′ (SEQ ID NO:5), 5′-AGGUAGAGU-3′, or 5′-AGGUAAGU-3′; the splice acceptor site can be 5′-GAUAUCCUGCAGG-3′ (SEQ ID NO:6), 5′-GGCUGCAGG-3′, or 5′-CCACAGC-3′; and the branch site can be 5′-UACUAAC-3′ or 5′-UACUUAUC-3′. The isolated RNA can be introduced into a cell for control of a gene function.
- The invention also provides a DNA template for the isolated RNA of the invention, an expression vector comprising the DNA, a cultivated cell comprising the isolated RNA or the DNA, an animal (e.g., a mammal such as a mouse) comprising the isolated RNA or the DNA, and a composition comprising the isolated RNA or the DNA.
- The invention further provides a method of producing an intron RNA. The method comprises cultivating the above-described cell to allow expression and/or release of the intron RNA. The released intron RNA can be left in the cell for control of a gene function, or be collected from the cell and used for generation of a DNA-RNA hybrid or delivery into another cell.
- Also within the scope of the invention is a method of modulating the function of a target gene in a cell. The method comprises introducing into a cell an effective amount of the isolated RNA or DNA of the invention. The intron RNA is then released in the cell, thereby modulating the function of a target gene.
- In another aspect, the invention features a composition comprising a chemokine (e.g., interleukin-2) and an isolated RNA or a DNA as described above. An effective amount of this composition can be administering into a cell (e.g., a mammalian cell or a cell infected by a virus) to modulate the function of a target gene. For example, an HIV-1-infected cell can be treated with a combination of interleukin-2 and an isolated RNA containing an intron RNA complementary to an HIV-1 genomic sequence. The intron RNA induces degradation of the HIV-1 genomic sequence or its derivatives, or prevent it from being translated into polypeptides.
- In still another aspect, the invention features a composition comprising one or more agents that induce RNA-mediated modulation of the functions of two or more target genes in a cell (e.g., a mammalian cell or a cell infected by a virus). A method of modulating the functions of genes in a cell by administering into the cell an effective amount of the composition is also within the scope of the invention. For example, when a cell is infected by HIV-1, it can be treated with one or more DNA-RNA hybrids or exogenous intron RNAs that cause degradation of HIV-1 RNAs, cellular RNAs such as Naf1β, Nb2HP, and Tax1BP RNAs, or their derivatives, or prevent these RNAs from being translated into polypeptides.
- The present invention provides compositions and methods for treating human diseases. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and the accompanying drawings, and from the claims.
-
FIG. 1 depicts a novel strategy for producing desired RNA molecules in cells through RNA splicing. -
FIG. 2 depicts generation of antisense RNAs by spliceosome cleavage of retroviral (e.g., LTR) promoter-mediated precursor transcripts. -
FIG. 3 depicts generation of sense and antisense siRNAs by spliceosome cleavage of viral (e.g., CMV or AMV) promoter-mediated precursor transcripts. -
FIG. 4 depicts generation of hairpin RNAs by spliceosome cleavage of Pol II (e.g., TRE or Tet response element) promoter-mediated precursor transcripts. -
FIG. 5 depicts microscopic results, showing interference with green fluorescent protein (eGFP) expression in rat neuronal stem cells by various SpRNAi constructs. -
FIG. 6 depicts Western blot results, showing interference with green fluorescent protein (eGFP) expression in rat neuronal stem cells by various SpRNAi constructs. -
FIG. 7 depicts Western blot results, showing interference with integrin β1 (ITGb1) expression in human prostatic cancer LNCaP cells by various SpRNAi constructs. -
FIGS. 8A-B depict Northern blot analysis of SpRNAi-induced cellular gene silencing against HIV-1 infection (n=3). -
FIG. 9 depicts potential differences between traditional PTGS/RNAi and SpRNAi phenomena. -
FIGS. 10A-B depict experimental evidence for generation of D-RNAi-induced miRNA. -
FIGS. 11A-C depict in vivo gene silencing by anti-β-catenin D-RNAi in embryonic chicken. -
FIG. 12 depicts in vivo gene silencing by anti-tyr D-RNAi in mouse. - This invention relates to RNA-mediated gene modulation.
FIG. 1 shows an example of a novel strategy for producing a desired RNA molecule in a cell after RNA splicing event occurs. The desired RNA molecule, like a natural intron, is flanked by an RNA splicing donor and an acceptor site. The DNA template for the desired RNA is inserted into a gene, which is expressed by type-II RNA polymerase (Pol II) transcription machinery under the control of either Pol II or viral RNA promoter. Upon intracellular transcription, the transcript so produced is subjected to RNA splicing and/or processing events, thereby releasing the desired RNA molecule. In certain cases, the desired RNA molecule is an antisense RNA that serves as an antisense oligonucleotide probe for antisense gene therapy. In other cases, the desired RNA molecule is translated into a polypeptide that is useful in gene replacement therapy. The desired RNA molecule can also consist of small antisense and sense RNA fragments to function as double-stranded siRNA for RNAi induction. Moreover, the desired RNA molecule can be a small hairpin-like RNA capable of causing RNAi-associated gene silencing phenomena. In addition, the desired RNA molecule can also be a ribozyme. All of the above desired RNA molecules are produced by intracellular splicing events and therefore named as “SpRNAi” for convenience. - Accordingly, the invention features an isolated RNA comprising an intron RNA that is released in a cell, thereby modulating the function of a target gene. An “isolated RNA” is a ribonucleic acid the structure of which is not identical to that of any naturally occurring ribonucleic acid or to that of any fragment of a naturally occurring ribonucleic acid. A “function of a target gene” refers to the capability of the target gene to be transcribed into an RNA, the capability of the RNA to be stabilized, processed (e.g., through splicing), reverse transcribed or translated, and the capability of the RNA to play its normal role, e.g., serving as a tRNA and rRNA.
- RNA splicing is a process that removes introns and joins exons in a primary transcript. The structures of intron RNAs are well known in the art. An intron usually contains signal sequences for splicing. For example, most introns start from the sequence GU and end with the sequence AG (in the 5′ to 3′ direction), which are referred to as the splice donor and splice acceptor site, respectively. In addition, an intron has a branch site between the donor and the acceptor site. The branch site contains an A residue (branch point), which is conserved in all genes. In many cases, the exon sequence is (A/C)AG at the 5′-exon-intron junction, and is G at the 3′-exon-intron junction. The fourth element is a poly-pyrimidine tract located between the branch site and the acceptor site.
- In an isolated RNA of the invention, the splice donor site may contain 5′-GUA(A/-)GAG(G/U)-3′, the splice acceptor site may contain 5′-G(A/U/-)(U/G)(C/G)C(U/C)(G/A)CAG-3′, a branch site may contain 5′-UACU(A/U)A(C/U)(-/C)-3′, and a poly-pyrimidine tract may contain 5′-(U(C/U))1-3(C/-)U7-12C(C/-)-3′ or 5′-(UC)7-12NCUAG(G/-)-3′. Functionally equivalents of these sequences (e.g., sequences containing modified nucleotides) are included in the invention. The intron RNA serves as or is farther processed to become, e.g., an RNA encoding a polypeptide, or an antisense RNA, short-temporary RNA (stRNA), microRNA (miRNA), small-interfering RNA (siRNA), short-hairpin RNA (shRNA), long deoxyribonucleotide-containing RNA (D-RNA), or ribozyme RNA, each of which may be in either sense or antisense orientation. Design of antisense RNA, stRNA, miRNA, siRNA, shRNA, D-RNA and ribozyme RNA is well known in the art. The intron RNA region homologous or complementary to its target gene ranges from 14 to 2,000 nucleotides, most preferably between 19 and 500 nucleotides. The intron RNA may be 35-100% (i.e., any integral between and including 35 and 100) identical or complementary to its target gene. The preferred homology or complementarity is 35-65% and more preferably 41-49% for an shRNA, 40-100% and more preferably 90-100% for a sense or antisense RNA. The length of an siRNA/miRNA/shRNA may be 16-38 nucleotides, and preferably 19-25 nucleotides. Additionally, there may be one or more linker sequences, e.g., between the donor and the acceptor site and the antisense RNA, stRNA, siRNA, shRNA, D-RNA or ribozyme RNA sequence. The isolated RNA may further contain exons encoding a polypeptide for co-expression with the intron RNA. The polypeptide may be a normal protein, a missing protein, a dominant-negative protein, or a protein marker such as a fluorescent protein, luciferase, or lac-Z.
- An isolated RNA of the invention can be chemically synthesized or produced by transcription from a DNA template in vitro and in vivo. The template DNA can be cloned into an expression vector according the methods well known in the art. Examples of such vectors include, but are not limited to, plasmids, cosmids, phagemids, yeast artificial chromosome, retroviral vectors, lentiviral vectors, lambda vector, adenoviral (AMV) vector, adeno-associated viral (AAV) vector, hepatitis virus (HBV)-modified vector, and cytomegalovirus (CMV)-related viral vector.
- The isolated RNA, DNA template, and expression vector described above can be introduced into a cultured cell or a subject (e.g., an animal or a human) using methods commonly employed in the art such as transfection, infection, electroporation, micro-injection, and gene-gun penetration. To help with the delivery into a cell, the isolated RNA, DNA template, and expression vector may be formulated into a composition. The intron RNA, once expressed and/or released in the cell, can modulate the function of a target gene, for example, inhibit a cancer-related gene, potential viral gene, and microbe-related gene. Therefore, this method is useful for treating and preventing diseases such as cancer and viral or microbial infection.
- In one in vivo approach, a composition is suspended in a pharmaceutically acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
- The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the subject's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100.0 μg/kg. Wide variations in the needed dosage are to be expected in view of the variety of compounds available and the different efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
- Alternatively, the composition may be delivered to the subject, for example, by use of polymeric, biodegradable microparticle or microcapsule delivery devices known in the art.
- Another way to achieve uptake of the nucleic acid is to use liposomes, prepared according to standard methods. The vectors can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies. Alternatively, one can prepare a molecular conjugate composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to a receptor on target cells (Cristiano et al. (1995) J. Mol. Med. 73: 479). Furthermore, tissue specific targeting can be achieved by use of tissue-specific transcriptional regulatory elements (TRE) which are known in the art. Delivery of naked nucleic acids (i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site is another means to achieve in vivo expression.
- An “effective amount” is an amount of the compound or composition that is capable of producing a medically desirable result (e.g., a decreased expression level of a cancer-related gene, potential viral gene, or microbe-related gene) in a treated subject.
- In particular, an animal comprising an isolated RNA or a DNA of the invention can be produced according to the methods described above or any other methods known in the art. For example, a “knock-out animal” may be generated in which a target gene is partially (e.g., only in some tissues) or completely inhibited. The animal can be a farm animal such as a pig, goat, sheep, cow, horse and rabbit, a rodent such as a rat, guinea pig, and mouse, or a non-human primate such as a baboon, monkey, and chimpanzee.
- These animals of the invention can be used as disease models. In particular, these animals can be used to identify a compound or composition effective for treatment or prevention of a disease. Compounds or compositions can be identified by administering a test compound or composition to a model animal or by contacting the test compound or composition with an organ, a tissue or cells derived from a model animal. Effects of the test compound or composition on the disease of the animal, organ, tissue or cells are evaluated. Test compounds or compositions that palliate the disease symptoms can be effective for treatment or prevention of the disease.
- A second aspect of the invention is based on the discovery that the combination of interleukin-2 and a viral RNA-antisense DNA hybrid significantly reduced human immunodeficiency virus-1 (HIV-1) subtype B gene activity. Consequently, the invention features a composition comprising a chemokine and an isolated RNA or a DNA of the invention. The isolated RNA or DNA allows an intron RNA to be released in a cell, thereby modulating the function of a target gene. Examples of chemokines include, but are not limited to, interleukin-2 (IL-2), interleukin-10 (IL-10), interleukin-17 (IL-17), tumor narcosis factor-α(TNF-α), and tumor narcosis factor-β(TNF-β). The intron RNA may contain, e.g., an antisense RNA, stRNA, miRNA, siRNA, shRNA, D-RNA, or ribozyme RNA. The composition can be administering into a cell according to the methods described above for modulating the function of a target gene in a cell, e.g., inducing degradation of an HIV-1 genomic sequence or preventing an HIV-1 genomic sequence from being translated into a polypeptide in an HIV-1 infected cell.
- It was also found that SpRNAi-induced silencing of cellular genes Naf1β, Nb2HP and Tax1BP prevents HIV-1 type B infection. The invention therefore provides a composition comprising one or more agents (e.g., an antisense RNA, stRNA, miRNA, siRNA, shRNA, D-RNA, SpRNAi, ribozyme RNA, or a combination thereof) that induce RNA-mediated modulation of the functions of two or more target genes in a cell. The composition can be administering into a cell according to the methods described above for control of the functions of genes.
- Applications of the present invention include, without limitation, therapy by suppression of cancer-related genes, vaccination against potential viral genes, treatment of microbe-related genes, research of candidate molecular pathways with systematic knockout/knockdown of involved molecules, and high-throughput screening of gene functions based on microarray analysis. The present invention can also be used as a tool for studying gene function under physiological and therapeutical conditions, providing compositions and methods for altering the characteristics of eukaryotic cells such as cancerous, virus-infected, microbe-infected, physiologically diseased, genetically mutated, and pathogenic cells.
- The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent.
- Rat neuronal stem cell clones AP31 and PZ5a were primary-cultured and maintained as described by Palmer et al. (1999) J. Neuroscience 19: 8487-8497. The cells were grown on polyornathine/laminin-coated dishes in DMEM/F-12 (1:1; high glucose) medium containing 1 mM·L-glutamine supplemented with 1×N2 supplements (Gibco/BRL, Gaithersburg, Md.) and 20 ng/ml FGF-2 (Invitrogen, Carlsbad, Calif.) without serum at 37° C. under 5% CO2. For long-term primary cultures, 75% of the medium was replaced with new growth medium every 48 h. Cultures were passaged at ˜80% confluency by exposing the cells to trypsin-EDTA solution (Irvine Scientific) for 1 min and rinsing them once with DMEM/F-12. Detached cells were replated at 1:10 dilution in fresh growth medium supplemented with 30% (v/v) conditioned medium which had been exposed to cells for 24 h before passaging. Human prostatic cancer LNCaP cells were obtained from American Type Culture Collection (ATCC, Rockville, Md.) and grown in RPMI 1640 medium supplemented with 10% fetal bovine serum with 100 μg/ml gentamycin at 37° C. under 10% CO2. The LNCaP culture was passaged at ˜80% confluency by exposing cells to trypsin-EDTA solution for 1 min and rinsing them once with RPMI, and detached cells were replated at 1:10 dilution in fresh growth medium. After a 48-hour incubation period, RNA was isolated from the cells using RNeasy spin columns (Qiagen, Valencia, Calif.), fractionated on a 1% formaldehyde-agarose gel, and transferred onto nylon membranes. The genomic DNA was also isolated using apoptotic DNA ladder kit (Roche Biochemicals, Indianapolis, Ind.) and assessed by 2% agarose gel electrophoresis, while cell growth and morphology were examined using microscopy and cell counting.
- Synthetic nucleic acid sequences used for generation of three different SpRNAi introns containing either sense, antisense or hairpin eGFP insert were as follows: N1-sense, 5′-pGTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA GA-3′ (SEQ ID NO:7); N1-antisense, 5′-pCGCGTCTTGA AGAAGATGGT GCGCTCCTGC GATCGGATCC TCTTAC-3′ (SEQ ID NO:8); N2-sense, 5′-pGTAAGAGGAT CCGATCGCTT GAAGAAGATG GTGCGCTCCT GA-3′ (SEQ ID NO:9); N2-antisense, 5′-pCGCGTCAGGA GCGCACCATC TTCTTCAAGC GATCGGATCC TCTTAC-3′ (SEQ ID NO:10); N3-sense, 5′-pGTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA GTTAACTTGA AGAAGATGGT GCGCTCCTGA-3′ (SEQ ID NO:11); N3-antisense, 5′-pCGCGTCAGGA GCGCACCATC TTCTTCAAGT TAACTTGAAG AAGATGGTGC GCTCCTGCGA TCGGATCCTC TTAC-3′ (SEQ ID NO:12); N4-sense, 5′-pCGCGTTACTA ACTGGTACCT CTTCTTTTTT TTTTTGATAT CCTGCAG-3′ (SEQ ID NO:13); N4-antisense, 5′-pGTCCTGCAGG ATATCAAAAA AAAAAGAAGA GGTACCAGTT AGTAA-3′ (SEQ ID NO:14). Additionally, two exon fragments were generated by DraII restriction enzyme cleavage of red fluorescent rGFP gene at nucleotide (nt) 208, and the 5′ fragment was further blunt-ended using T4 DNA polymerase. The rGFP refers to a new red-fluorescin chromoprotein generated by insertion of an additional aspartate at amino acid (aa) 69 of HcRed1 chromoprotein from Heteractis crispa. (Gurskaya et al. (2001) FEBS Letters 507: 16-20), developing less aggregate and almost twice intense far-red fluorescent emission at ˜570-nm wavelength. This mutated rGFP gene sequence was cloned into pHcRed1-N1/1 plasmid vector (BD Biosciences) and propagated in E. coli DH5α in LB medium containing 50 μg/ml kanamycin (Sigma). The pHcRed1-N1/1 plasmid was cleaved with XhoI and XbaI restriction enzymes. A 769-bp rGFP fragment and a 3,934-bp empty plasmid were purified separately from a 2% agarose gel after electrophoresis.
- Hybridization of N1-sense to N1-antisense, N2-sense to N2-antisense, N3-sense to N3-antisense, and N4-sense to N4-antisense was separately performed by heating each mixture of complementary sense and antisense (1:1) sequences to 94° C. for 2 min and then 70° C. for 10 min in 1×PCR buffer (e.g., 50 mM Tris-HCl, pH 9.2 at 25° C., 16 mM (NH4)2SO4, 1.75 mM MgCl2). Subsequently, ligation of the N1, N2 or N3 hybrid to the N4 hybrid was performed by gradually cooling a mixture of N1-N4, N2-N4 or N3-N4 (1:1) hybrids from 50° C. to 10° C. over a period of 1 h, and then T4 ligase and buffer (Roche) were mixed with the mixture for 12 h at 12° C. so as to obtain introns for linking to exons with proper ends. After the rGFP exon fragments were added into the reaction (1:1:1), T4 ligase and buffer were adjusted accordingly for continued ligation for another 12 h at 12° C. For cloning the right sized recombinant rGFP gene, 10 ng of the ligated nucleotide sequences were amplified by PCR with rGFP-
specific primers 5′-dCTCGAGCATG GTGAGCGGCC TGCTGAA-3′ (SEQ ID NO:15) and 5′-dTCTAGAAGTT GGCCTTCTCG GGCAGGT-3′ (SEQ ID NO:16) at 94° C., 1 min; 52° C., 1 min; and then 68° C., 2 min for 30 cycles. The PCR products were fractionated on a 2% agarose gel, and a ˜900-bp nucleotide sequence was extracted and purified using a gel extraction kit (Qiagen). The composition of this ˜900 bp SpRNAi-eGFP-containing rGFP gene was confirmed by sequencing. - Since the recombinant gene possesses an XhoI and an XbaI restriction site at its 5′- and 3′-end, respectively, it can be easily cloned into a vector with ends complementary to the XhoI and XbaI sites. The vector can be an expression vector, e.g., a plasmid, cosmid, phagmid, yeast artificial chromosome, or viral vector. Moreover, since the insert within the intron is flanked by a PvuI and an MluI restriction site at its 5′- and 3′-end, respectively, the insert can be removed and replaced with another insert with ends complementary to the PvuI and MluI sites. The insert sequence can be homologous or complementary to a gene fragment such as a fluorescent protein gene, luciferase gene, lac-Z gene, plant gene, viral genome, bacterial gene, animal gene, and human oncogene. The homology and/or complementarity ranges from about 30˜400%, more preferably 35˜49% for a hairpin-shRNA insert and 90˜100% for both sense-siRNA and antisense-siRNA inserts.
- For cloning into plasmids, the SpRNAi-recombinant rGFP gene and the linearized 3,934-bp empty pHcRed1-N1/1 plasmid were mixed at 1:16 (w/w) ratio. The mixture was cooled from 65° C. to 15° C. over a period of 50 min, and then T4 ligase and buffer were added into the mixture for ligation at 12° C. for 12 h. A so formed SpRNAi-recombinant rGFP-expressing plasmid vector was propagated in E. coli DH5α in LB medium containing 50 μg/ml kanamycin. A positive clone was confirmed by PCR with rGFP-specific primers SEQ ID NO:15 and SEQ ID NO:16 at 94° C., 1 min and then 68° C., 2 min for 30 cycles and subsequent sequencing. For cloning into viral vectors, the same ligation procedure was performed except that an XhoI/XbaI-linearized pLNCX2 retroviral vector (BD Biosciences) was used. The eGFP insert within the SpRNAi intron was removed and replaced with various integrin β1-specific insert sequences with ends complementary to the PvuI and MluI sites.
- Synthetic nucleic acid sequences used for generation of various SpRNAi introns containing either sense, antisense or hairpin integrin β1 insert were as follows P1-sense, 5′-pCGCAAGCAGG GCCAAATTGT GGGTA-3′ (SEQ ID NO:17); P1-antisense, 5′-pTAGCACCCAC AATTTGGCCC TGCTTGTGCG C-3′ (SEQ ID NO:18); P2-sense, 5′-pCGACCCACAA TTTGGCCCTG CTTGA-3′ (SEQ ID NO:19); P2-antisense, 5′-pTAGCCAAGCA GGGCCAAATT GTGGGTTGCG C-3′ (SEQ ID NO:20); P3-sense, 5′-pCGCAAGCAGG GCCAAATTGT GGGTTTAAAC CCACAATTTG GCCCTGCTTG A-3′ (SEQ ID NO:21); P3-antisense, 5′-pTAGCACCCAC AATTTGGCCC TGCTTGAATT CAAGCAGGGC CAAATTGTGG GTTGCGC (SEQ ID NO:22). These inserts were designed using Gene Runner software v3.0 (Hastings, Calif.) and formed by hybridization of P1-sense to P1-antisense, P2-sense to P2-antisense and P3-sense to P3-antisense for targeting nt 244˜265 of the integrin β1 gene (GenBank Access No. NM 002211.2). The SpRNAi-containing rGFP-expressing retroviral vector was propagated in E. coli DH5α in LB medium containing 100 μg/m ampcillin (Sigma). A packaging cell line PT67 (BD Biosciences) was also used for producing infectious, replication-incompetent viruses. Transfected PT67 cells were grown in DMEM medium supplemented with 10% fetal bovine serum with 4 mM L-glutamine, 1 mM sodium pyruvate, 100 μg/ml streptomycin sulfate and 50 mg/ml neomycin (Sigma) at 37° C. under 5% CO2. The titer of the virus produced by PT67 cells was determined to be at least 106 cfu/ml before transfection.
- RNA (20 μg total RNA or 2 μg poly[A+] RNA) was fractionated on 1% formaldehyde-agarose gels and transferred onto nylon membranes (Schleicher & Schuell, Keene, N. H.). A synthetic 75-bp probe (5′-dCCTGGCCCCC TGCTGCGAGT ACGGCAGCAG GACGTAAGAG GATCCGATCG CAGGAGCGCA CCATCTTCTT CAAGT-3′ (SEQ ID NO:23)) targeting the junction region between rGFP and the hairpin eGFP-insert was labeled with the Prime-It II kit (Stratagene, La Jolla, Calif.) by random primer extension in the presence of [32P]-dATP (>3000 Ci/mM, Amersham International, Arlington Heights, Ill.), and purified using 30 bp-cutoff Micro Bio-Spin chromatography columns (Bio-Rad, Hercules, Calif.). Hybridization was carried out in a mixture of 50% freshly deionized formamide (pH 7.0), 5×Denhardt's solution, 0.5% SDS, 4×SSPE and 250 mg/mL denatured salmon sperm DNAs (18 h, 42° C.). Membranes were sequentially washed twice in 2×SSC, 0.1% SDS (15 min, 25° C.), and once in 0.2×SSC, 0.1% SDS (15 min, 25° C.) before autoradiography.
- For interference with eGFP expression, rat neuronal stem cells were transfected with SpRNAi-recombinant rGFP plasmids encoding either a sense, antisense or hairpin eGFP insert using Fugene reagent (Roche). Plasmids containing insert-free rGFP gene and SpRNAi-recombinant rGFP gene with an insert against HIV-gag p24 were used as negative controls. Cell morphology and fluorescent images were photographed at 0-, 24- and 48-hour time points after transfection. At the 48-h incubation time point, the rGFP-positive cells were sorted by flow cytometry and collected for Western blot analysis. For interference with integrin β1 expression, LNCaP cells were transfected with pLNCX2 retroviral vectors containing various SpRNAi-recombinant rGFP genes against nt 244˜265 of integrin β1 using the Fugene reagent. The transfection rate of pLNCX2 retroviral vector into LNCaP cells was determined to be about 20%, while the pLNCX2 virus was less infectious to LNCaP cells. The same analyses were performed as aforementioned.
- For immunoblotting, cells were rinsed with ice-cold PBS after the growth medium was removed, and then treated with the CelLytic-M lysis/extraction reagent (Sigma Chemical, St. Louis, Mo.) supplemented with protease inhibitors, Leupeptin, TLCK, TAME and PMSF following manufacturer's recommendations. The cells were incubated at room temperature on a shaker for 15 min, scraped into microtubes, and centrifuged for 5 min at 12,000×g to pellet the cell debris. Protein-containing cell lysate was collected and stored at −70° C. until use. Protein concentrations were determined as described (Bradford (1976) Anal. Biochem. 72: 248-254) using SOFTmax software package on an E-max microplate reader (Molecular Devices, Sunnyvale, Calif.). 30 μg of cell lysate was added into SDS-PAGE sample buffer either with (reduced) or without (unreduced) 50 mM DTT, and boiled for 3 min before loading onto 8% polyacrylamide gels, while the reference lane was loaded with 2˜3 μl molecular weight markers (BioRad). SDS-polyacrylamide gel electrophoresis was performed according to the standard protocols (Sambrook and Russell, Molecular Cloning, 3rd Ed., (2001) Cold Spring Harbor Laboratory Press: New York). Protein fractions were electroblotted onto a nitrocellulose membrane, blocked with Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) for 1˜2 h at room temperature. GFP expression was assessed using primary antibodies directed against eGFP (1:5,000; JL-8, BD Biosciences, Palo Alto, Calif.) or rGFP (1:10,000; BD Biosciences) overnight at 4° C. The blot was then rinsed 3 times with TBS-T and exposed to a secondary antibody, goat anti-mouse IgG conjugate with Alexa Fluor 680 reactive dye (1:2,000; Molecular Probes), for 1 h at room temperature. After three more TBS-T rinses, scanning and image analysis were performed using Li-Cor Odyssey Infrared Imager and Odyssey Software v.10 (Li-Cor). For integrin β1 analysis, the same procedure was performed except that primary antibodies directed against integrin β1 (1:2,000; LM534, Chemicon, Temecula, Calif.) were used.
- The RNA-polymerase cycling reaction (RNA-PCR) procedure can be modified to synthesize mRNA-aDNA and/or mDNA-aRNA hybrids (Lin et al. (1999) Nucleic Acids Res. 27, 4585-4589) from an SpRNAi-recombinant gene, expression-competent vector template or transcriptome source. As an example of using an SpRNAi-recombinant gene source, an SpRNAi-sense HIV recombinant gene containing a sequence homologues to HIV-1 genome from +2113 to +2453 bases was generated following a procedure similar to
Section 2 above. The RNA product (10˜50 μg) of the SpRNAi-sense HIV recombinant gene were transcribed in about 106 transfected cells, isolated using RNeasy columns (Qiagen), and then hybridized to its pre-synthesized complementary DNA (cDNA) by heating and then cooling the mixture from 65° C. to 15° C. over a period of 50 min. Transfection was performed following the same procedure shown inSection 5 above. - RNA splicing/processing-directed gene silencing was tested using an artificial recombinant gene, SpRNAi-rGFP (
FIG. 1 ). A DNA template for a splicing-competent intron (SpRNAi) was inserted into an intron-free red fluorescin gene (rGFP), providing splicing-directed gene silencing effects through pre-mRNA splicing and some unknown processing mechanisms. Although a model of gene silencing through pre-mRNA splicing is shown here, the same principle can be used for the design of gene silencing inserts working through other pre-RNA processing, e.g., pre-ribosomal RNA (pre-rRNA)-processing, which is mainly mediated by type-I RNA polymerase (Pol I) transcription machinery. The splicing-competent intron is flanked by a donor (DS) and an acceptor (AS) splicing site, and contains at least one gene homologue insert, branch point (BrP) and poly-pyrimidine tract (PPT) inbetween the DS-AS sites for interacting with spliceosome machinery. Using low stringency Northern blotting (mid-bottom ofFIG. 1 ), 15˜45 bp intron-insert fragments were seen to be released from the SpRNAi-rGFP gene transcript (left), rather than an intron-free rGFP (middle) or a defective SpRNAi-rGFP (right) RNA without a functional splice donor site, while the spliced exons were linked to form a mature RNA for rGFP protein synthesis. The “?” mark inFIG. 1 indicates an unknown mechanism for processing of a ˜120-bp intron, resulting in small interfering intron-insert fragments. Short sense, short antisense and hairpin constructs of some gene homologue inserts were successfully tested for inducing specific gene silencing in various cell types. - As shown in
FIG. 1 , DNA templates for splicing-competent introns (SpRNAi) were synthesized and inserted into an intron-free red fluorescin gene (rGFP) that was mutated from the HcRed1 chromoprotein of Heteractis crispa. Since the inserted intron disrupted the functional fluorescin structure of the rGFP protein, occurrence of intron splicing and rGFP-mRNA maturation was indicated by the reappearance of red fluorescent light emission at the 570-nm wavelength in a transfected cell. Construction of SpRNAi was based on the natural structure of a pre-messenger RNA intron, consisting of spliceosome-dependent nucleotide components, such as donor and acceptor splicing sites in both ends for precise cleavage, branch point domain for splicing recognition, poly-pyrimidine tract for spliceosome interaction, linkers for connection of each major components and some artificially added multiple restriction/cloning sites for cloning of inserts. Based on prior studies, the donor splicing site is an oligonucleotide sequence either containing or homologous to 5′-exon-AG-(splicing point)-GTA(A/-)GAG(G/T)-3′ (SEQ ID NO:24), e.g., 5′-AG GTAAGAGGAT-3′ (SEQ ID NO:25), 5′-AG GTAAGAGT-3′ (SEQ ID NO:26), 5′-AG GTAGAGT-3′, 5′-AG GTAAGT-3′ and so on. The acceptor splicing site is an oligonucleotide sequence either containing or homologous to 5′-G(W/-)(T/G)(C/G)C(T/C)(G/A)CAG-(splicing point)-G/C-exon-3′ (while W is a pyrimidine, i.e., A or T) (SEQ ID NO:27), e.g., 5′-GATATCCTGCAG G-3′ (SEQ ID NO:28), 5′-GGCTGCAG G-3′, 5′-CCACAG C-3′ and so on. The branch point is an “A” residue located within a sequence homologous to 5′-TACT(A/T)A*(C/T)(-/C)-3′ (while the symbol “*” marks the branch site), e.g., 5′-TACTAAC-3′, 5′-TACTTATC-3′ and so on. The poly-pyrimidine tract is a high T and/or C content oligonucleotide sequence homologous to 5′-(TY)m(C/-)(T)nC(C/-)-3′ or 5′-(TC)nNCTAG(G/-)-3′ (while Y is a C or T). The symbols “m” and “n” indicate the numbers of repeats, preferably, m=1˜3 and n=7˜12. For all the above splicing components, the deoxythymidine (T) in a DNA template is replaced by uridine (U) after transcription. - To test the function of a spliced intron, various inserts were cloned into SpRNAi through multiple restriction/cloning sites, e.g., those for AatII, AccI, AflII/III, AgeI ApaI/LI, AseI, Asp718I, BamHI, BbeI, BclI/II, BglII, BsmI, Bsp120I, BspHI/LU11I/120I, BsrI/BI/GI, BssHII/SI, BstBI/U1/XI, ClaI, Csp6I, DpnI, DraI/II, EagI, Ecl136II, EcoRI/RII/47III, EheI, FspI, HaeIII, HhaI, HinPI, HindIII, HinfI, HpaI/II, KasI, KpnI, MaeII/III, MfeI, MluI, MscI, MseI, NaeI, NarI, NcoI, NdeI, NgoMI, NotI, NruI, NsiI, Pm1I, Ppu10I, PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI and/or XmaI endonucleases. These inserts are DNA templates for aberrant RNAs, e.g., short-temporary RNA (stRNA), small-interfering RNA (siRNA), short-hairpin RNA (shRNA), long deoxyribonucleotide-containing RNA (D-RNA) and potentially ribozyme RNA in either sense or antisense orientation. As demonstrated in the examples below, the gene silencing effect of a hairpin-RNA-containing SpRNAi is stronger than that of a sense- and antisense-RNA-containing SpRNAi, showing an average of >80% knockdown specificity to all targeted gene products. Such knockdown specificity is mainly determined by the homologous or complementary region of an insert to the targeted gene transcript. For example, the tested hairpin-SpRNAi insert had about 40˜42% homology and another 40˜42% complementarity to the targeted gene domain, with-in-between of which an A/T-rich linker sequence filled in the
rest 8˜10% space. For the less potent sense- and antisense-SpRNAi inserts, although the homology or complementarity can be increased up to 100%, an average of 40˜50% knockdown efficacy was detected in most of the transfection tests. Thus, different types of SpRNAi inserts and/or the combination thereof can be used to manipulate specific gene expression levels in cells. - For the convenience of gene delivery and activation in cells, SpRNAi-containing genes were cloned into an expression-competent vector, e.g., plasmid, cosmid, phagmid, yeast artificial chromosome, viral vector and so on. As shown in
FIGS. 1-4 , the vectors can contain at least one viral or type-II RNA polymerase (Pol II) promoter or both for the expressing of the SpRNAi-gene in eukaryotic cells, a Kozak consensus translation initiation site to increase translation efficiency in eukaryotic cells, SV40 polyadenylation signals downstream of the SpRNAi-gene for processing of the 3′-end gene transcript, a pUC origin of replication for propagation in prokaryotic cells, at least two multiple restriction/cloning sites for cloning of the SpRNAi-gene, an optional SV40 origin for replication in mammalian cells expressing the SV40 T antigen and an optional SV40 early promoter for expressing an antibiotic resistance gene in replication-competent prokaryotic cells. The expression of antibiotic resistance genes was used as a selective marker for searching of successfully transfected or infected clones that are resistance to the antibiotics such as penicillin G, ampcillin, neomycin, paromycin, kanamycin, streptomycin, erythromycin, spectromycin, phophomycin, tetracycline, rifapicin, amphotericin B, gentamicin, chloramphenicol, cephalothin, tylosin and the combination thereof. The vector was therefore stable enough to be introduced into a cell(s), tissue or animal body using a highly efficient gene delivery method, e.g., liposomal transfection, chemical transfection, chemical transformation, electroporation, infection, micro-injection and gene-gun penetration. - As shown in
FIG. 5 , transfection of the plasmids described inSections 2 and 3 (containing various SpRNAi-rGFP recombinant genes against the expression of a commercially available Aequorea victoria green fluorescent protein (eGFP)) was found to be successful in both expression of rGFP (red) and silencing eGFP (green). The use of eGFP-positive rat neuronal stem cell clones provided an excellent visual aid to measure the silencing effects of various SpRNAi inserts. Rat neuronal stem cell clones AP31 and PZ5a were primary-cultured and maintained as described inSection 1. 24-h after transfection, almost the same amount of total cell number and eGFP-positive cell population were well seeded and very limited apoptotic or differentiated cells occurred. Silencing of eGFP emission at the 518-nm wavelength was detected 36˜48 hours after transfection, indicating a potential onset timing required for the release of small interfering inserts from SpRNAi-rGFP gene transcripts by spliceosome machinery. Since all successfully transfected cells displayed red fluorescent emission at about 570-nm wavelength, the gene silencing effect was traced by measuring relative light intensity of the green fluorescent emission in the red fluorescent cells, showing a knockdown potency of hairpin-eGFP>>sense-eGFP˜antisense-eGFP>>hairpin-HIV p24 (negative control) insert. - As shown in
FIG. 6 , knockdown levels of eGFP protein in rat neuronal stem cell clones AP31 and PZ5a by various SpRNAi inserts were measured on an unreduced 6% SDS-polyacrylamide gel. To normalize the loading amounts of transfected cellular proteins, rGFP protein levels (˜30 kDa, red bars) were adjusted to be comparatively equal, representing an average expression range of 82˜100% intensity (Y axis). The eGFP levels (27 kDa, green bars) were found to be reduced by transfection of SpRNAi-rGFP genes containing sense-eGFP (43.6%), antisense-eGFP (49.8%) or hairpin-eGFP (19.0%) inserts, but not that of intron-free rGFP gene (blank control) or SpRNAi-rGFP gene containing hairpin-HIV p24 insert (negative control). These findings confirm the knockdown potency of hairpin-eGFP>>sense-eGFP˜antisense-eGFP>>hairpin-HIV p24 (negative control), and also demonstrate that only a gene insert which is either homologous or complementary (or partially homologous or complementary) to the targeted gene can elicit this gene-specific silencing effect. - As shown in
FIG. 7 , a similar splicing/processing-directed gene silencing phenomenon was seen in human cancerous LNCaP cells. Knockdown levels of integrin β1 (ITGb1) protein by various SpRNAi inserts, were measured on a reduced 8% SDS-polyacrylamide gel. The relative amounts of rGFP (black bars), ITGb1 (gray bars) and actin (white bars) were shown on a percentage scale (Y axis). The ITGb1 levels (29 kDa) were significantly reduced by transfection of SpRNAi-rGFP genes containing a sense-ITGb1 (37.3%), antisense-ITGb1 (48.1%) and hairpin-ITGb1 (13.5%) inserts, but not that of an intron-free rGFP gene (blank control) or SpRNAi-rGFP gene containing a hairpin-HIV p24 insert (negative control). Co-transfection of SpRNAi-rGFP genes containing sense- and antisense-ITGb1 inserts elicited a significant gene silencing effect (22.5%) and 10˜15% cell death, while that of SpRNAi-rGFP genes containing hairpin-ITGb1 and hairpin-p58/HHR23 inserts partially blocked the splicing-directed gene silencing effect and resulted in an average of 57.8% expression level. These findings indicate that the SpRNAi-induced gene silencing effects may work on a wide range of genes and cell types of interest. - The ex vivo transfection of a viral RNA-antisense DNA hybrid construct in conjunction with
interleukin 2 adjuvant therapy was found to silence an average of 99.8% human immunodeficiency virus-1 (HIV-1) subtype B gene activity through a novel posttranscriptional gene silencing mechanism, deoxyribonucleotidylated RNA interference (D-RNAi; Lin et al. (2001) supra). This combined therapy not only delivered a strong suppression effect on viral replication but also boosted the immunity and proliferation of non-infected CD4+ T lymphocytes. A normal T cell outgrowth effect was observed to achieve maximal 76.2% HIV-infected cell elimination after one-week of therapy. RNA-directed endoribonuclease activity was mildly increased up to 6.7% by the transfection, while no interferon-induced cytotoxicity was detected. The cellular genes corresponding to combinational therapy have been, further investigated by microarray analysis for AIDS prevention. Co-suppression of three microarray-identified target genes, Naf1β, Nb2 protein homologous to Wnt-6 and Tax1 binding protein was shown to prevent an average of 80.2% HIV-1b entry and infection in a primary CD4+ T cell model. These findings indicate an immediate therapy in both acute and chronic HIV-1 infections and also a potential vaccination useful for AIDS elimination. - In order to test the effectiveness of D-RNAi to inactivate HIV-1 replication, a viral RNA (vRNA)-antisense DNA (aDNA) hybrid construct was designed to silence an early-stage gene locus containing gag/pol/pro viral genes and p24 HIV-1 gene marker. The anti-gag/pol/pro transfection interferes with the integration of viral provirus into host chromosome and also prevents the activation of several viral genes, while the anti-p24 transfection provides a visual indicator for observing viral activity on an ELISA assay. The results showed that such strategy was effective in knocking out exogenous viral gene expression ex vivo in a CD4+ T lymphocyte extract model. Peripheral blood mononuclear cells (PBMC) extracted from patients were purified using CD4+-affinity immunomagnetic beads and grown in RPMI 1640 medium with 200 U/ml IL-2 adjuvant treatment for more than two weeks. A vRNA-aDNA hybrid probe containing partial HIV genomic sequence from +2113 to +2453 bases was generated using a pre-designed SpRNAi-recombinant gene (used as a control as described in previous sections) homologous to gag-p24 genes. After 96-h incubation, the expression activity of HIV-1 genome was measured by Northern blotting and found to be almost completely shut down in the D-RNAi hybrid transfection sets.
- The gene silencing effects of anti-HIV D-RNAi transfections in the acute phase AIDS patient T lymphocyte extracts were biostatistically significant (n=3, p<0.01). Pure HIV-1 provirus was shown as a viral genome sized about 9.7 kb on a formaldehyde-containing RNA electrophoresis gel. Samples of CD4+ Th lymphocyte RNA extracts from normal, non-infected persons were used as negative controls, while those from HIV-1 infected patients were used as positive controls. No significant gene silencing effect was detected in all controls or transfections of other constructs, including vDNA-aRNA hybrid of HIV-1b, aDNA only and vRNA-aDNA against HTLV-1 rather than HIV-1. In the acute phase (<2-week infection), treatment with 5 nM D-RNAi knocked out an average of 99.8% viral gene expression, whereas in the chronic phase (˜two-year infection), the same treatment knocked down only an average of 71.4% viral gene expression. Although higher RNase activities were found in chronic HIV-1+ T cells by microarray analysis, transfection of D-RNAi in higher concentration (more than 25 nM) can overcome this drug resistance. Unlike dsRNA, transfection of highly concentrated vRNA-aDNA hybrids did not cause significant interferon-induced cytotoxic effects, because the house-keeping gene, β-actin, was expressed normally in all sets of cells. Since the Northern blot method is able to detect HIV-1 gene transcript at the nanogram level, the above strong viral gene silencing effect suggests a very promising pharmaceutical and therapeutical potential for combinational administration of D-RNAi and IL-2 as antiviral therapy and/or vaccination.
- Northern blot analysis of SpRNAi-induced gene silencing effects on Naf1β, Nb2HP and Tax1BP was shown to prevent HIV-1 type B infection (
FIG. 8 ). The tested gene targets were selected through RNA-PCR microarray analysis of differential expression genes from the acute (one˜two weeks) and chronic (about two years) infected patients' primary T cells with or without 25 nM anti-HIV D-RNAi treatment (Lin et al. (2001) supra). The SpRNAi product concentrations in all treatments were normalized to 30 nM.FIG. 8B is a bar chart showing HIV-gag p24 ELISA results (white) in correlation with the treatment results demonstrated inFIG. 8A . - In view of CD4 function in IL-2 stimulation and T-cell growth and activation, CD4 may not be an ideal target for HIV prevention. However, the search for HIV-dependent cellular genes in vivo was hindered by the fact that infectivity of viruses and infection rate among different patients are usually different and lead to inconsistent results. Short-term ex vivo culture conditions can normalize infectivity and infection rate of HIV transmission in a more uniformed CD4+ T cell population. Microarray analysis based on such ex vivo conditions would be reliable for critical biomedical and genetic research of HIV-1 infection. Microarray studies identified differential gene profiles between HIV− and HIV+ T cells in the acute and chronic infection phases and provided many potential anti-HIV cellular gene targets for AIDS therapy and prevention. To functionally evaluate the usefulness of targeting cellular genes for HIV vaccination, three highly differentially expressed genes, Naf1β, Nb2 homologous protein to Wnt-6 (Nb2HP) and Tax1 binding protein (Tax1BP) were tested for inhibiting HIV-1 infection. Since each of these genes contributes to only a part of AIDS complications, knockdown of a single target gene failed to suppress HIV-1 infection, while combination of all three SpRNAi probes at the same total concentration showed a significant (80±10%) reduction in HIV-1b infection (
FIG. 8A , n=3, p<0.01). The ELISA results of HIV gag-p24 protein (FIG. 8B ) also correlated with the Northern blot data, showing an average of 77±5% reduction of gag-p24 expression. These findings indicate the feasibility of a novel strategy for retroviral vaccination using PTGS mechanisms against cellular target genes. - Two major phenomenal differences between PTGS/RNAi and SpRNAi mechanisms were found. First, the onset of SpRNAi effects takes a period of time more than 36-48 hours, which is longer than the timing needed for the onset of PTGS/RNAi (12-24 hours). Second, although the role of PTGS/RNAi-associated Dicer enzymes is unclear for the SpRNAi-directed gene silencing mechanism, several repair complementing antigens were found to be involved. Homologous recombination machinery involving nucleotide excision repair-related gene p58/HHR23 was found to play a potential role of Dicer in SpRNAi induction. The p58/HHR23 species that codes for XP-C repair-complementing proteins is a human homologue of yeast RAD23 derivatives, sharing an ubiquitin-like N-terminus. Based on its molecular similarity shared with RNA repairing-directed transcriptional regulation, the repair-complementing machinery indicates a novel mechanism of posttranscriptional gene silencing in addition to RNA interference.
- Homologous recombination between intracellular mRNAs and the RNA components of a D-RNAi agent construct probably accounted for its specific gene silencing effect (Lin et al. (2001) Current Cancer Drug Targets 1: 241-247). [P32]-labeled DNA component of a D-RNAi agent construct was found to be intact in a hybrid duplex during the effective period of a D-RNAi phenomenon, while the labeled RNA part was replaced by a cold homologue and degraded into small RNA oligoribonucleotides within a 3-day incubation period (
FIG. 10A ). It is possibly that the D-RNAi agent can facilitate the degradation of non-recombined parts of its mRNA homologue as shown inFIG. 10B . Alternatively, the newly recombined mRNA part of the D-RNAi agent may be further processed by intracellular Pol II and some unknown RNA excision machineries to generate miRNA-like molecules for long-term gene silencing. This is supported by the fact that both D-RNAi-derived small RNAs and Pol II RNA splicing-processed intron fragments have an average length of 15-45 nucleotides, which is comparable to the general sizes of Dicer-processed miRNA intermediates. Additionally, both kinds of small RNAs isolated by guanidinium-chloride ultracentrifugation can elicit strong, but short-term gene silencing effects to genes homologous to the small RNAs in cells, indicating the possible miRNA-related interfering property of these small RNAs. Since the small miRNA-like RNAs are constitutionally derived from the large templates of mRNA-cDNA or precursor mRNA-genomic DNA hybrids, the long-term effect of D-RNAi phenomena may be maintained by accumulation of sufficient small miRNA-like RNAs rather than the stability of small RNAs. This also explains the delayed initiation phase observed in the D-RNAi-induced gene silencing and intron splicing-mediated PTGS phenomena (Lin et al. (2001) Biochem. Biophys. Res. Commun. 281: 639-644; and Lin et al. (2003) Biochem. Biophys. Res. Commun., in press). - Previous studies (Zhang et al. (1994) Nature 372: 809-812; and Ghosh and Garcia-Blanco (2000) RNA 6: 1325-1334) have demonstrated that a coupled interaction between nascent Pol II pre-mRNA transcription and intron excision occurs within certain nuclear region proximal to genomic DNA (i.e., perichromatin fibrils), indicating a reasonable potential for D-RNAi-associated miRNA generation in cells. The spliced introns are not completely digested into monoribonucleotides for transcriptional recycling, as approximately 10˜30% of the introns are found in the cytoplasm with a moderate half-life (Nott et al. (2003) RNA 9: 607-617). In an effort to examine such a process, an artificial intron mimicking the natural structure of a pre-mRNA intron was constructed for evaluating splicing-directed small RNA generation (Lin et al. (2003) Biochem. Biophys. Res. Commun., in press). The splicing-competent artificial intron, SpRNAi, is flanked by a splice donor (DS) and acceptor (AS) site, and contains a branch-point domain (BrP), a poly-pyrimidine tract (PPT) and at least one intronic insert located in the 5′-proximal domain of the artificial intron. To ensure the accuracy of pre-mRNA splicing, the SpRNAi also contains a translation stop codon in its 3′-proximal region, which if present in a cytoplasmic mRNA, would signal the diversion of the defective pre-mRNA from a non-sense mRNA degradation (NMD) pathway. As shown by results from low stringency Northern blotting, the intracellular processing of a spliced intron into small RNA fragments was found to be highly efficient. The release of small 15˜45 nt RNA fragments was found to be only from the intron-containing gene transcripts, but not from an intronless mRNA or a splice-donor-defective pre-mRNA (a positive example of NMD). The small miRNA-like RNAs are able to trigger translation repression or sometimes RNA degradation depending on the degree of complementarity and homology with their targets. According to the variety and complexity of natural miRNA structures, there is no artificial means to produce intracellular miRNA-like molecules before the finding of this intron splicing-mediated gene silencing phenomenon. The process of such miRNA-like small interfering RNA generation is therefore different from that for the dsRNA-induced RNAi; however, the possible involvement of RNAi mechanisms cannot be ruled out in that some small RNAs might form siRNAs by complementary hybridization within a localized compartment.
- D-RNAi can be used as an effective strategy to silence specific target gene in vivo. β-catenin gene was selected as an example because its product plays a critical role in the biological development and ontogenesis. β-catenin is known to be involved in the growth control of skin and liver tissues in chicken embryos. As shown in
FIG. 11 , experimental results demonstrated that D-RNAi (mRNA-cDNA hybrid) agents were capable of inhibiting β-catenin gene expression in the liver and skin of developing chicken embryos. The anti-β-catenin D-RNAi molecules were generated against the central region (aa 306-644) of the β-catenin coding sequence (GenBank Access No. X87838) by RNA-PCR. Fertilized eggs were obtained from SPAFAS farm (Preston, Conn.) and incubated in a humidified incubator. Usingembryonic day 3 chicken embryos, a dose of 25 nM of the D-RNAi agent or reversal control hybrids of sense DNA-antisense RNA (sDNA-aRNA) was injected into the ventral body cavity, which is close to where the liver primordia would form (FIG. 11A ). The mRNA-cDNA or the control sDNA-aRNA hybrids were mixed with DOTAP® liposomal transfection reagent (Roche) at a ratio of 3:2. A 10% (v/v) fast green solution was added before injection as a dye indicator. The mixtures were injected into the ventral side near the liver primordia and below the heart using heat pulled capillary needles. After injection, the eggs were sealed with scotch tape and put back into the humidified incubator at 39-40° C. until day 12 when the embryos were removed, examined and photographed under a dissection microscope. While there were malformations, the embryos survived and there was no visible overt toxicity or overall perturbation of embryo development. The liver was the closest organ to the injection site and was most dramatically affected in its phenotypes. Other regions, particularly the skin, were also affected by the diffused D-RNAi agent. As shown inFIG. 11B , Northern blot hybridizations using RNA from dissected livers showed that β-catenin in the control livers remained expressed (lanes 4-6), whereas the level of β-catenin mRNA was decreased dramatically (lanes 1-3) after treatment with the D-RNAi agent directed against β-catenin. Controls used included liposome alone (lane 4) and of control sDNA-aRNA hybrids in similar concentrations (lanes 5 and 6). - After ten days of injection with the anti-β-catenin D-RNAi (mRNA-cDNA hybrid) agent, the embryonic chicken livers showed an enlarged and engorged first lobe, but the size of the second and third lobes of the livers were dramatically decreased (
FIG. 11C ). Histological sections of normal livers showed hepatic cords and sinusoidal space with few blood cells. In anti-β-catenin D-RNAi-treated embryos, the general architecture of the hepatic cells inlobes lobe 1. The endothelium development appeared to be defective and blood leaked outside of the blood vessels. Abnormal types of hematopoietic cells were also observed between hepatocytes, particularly dominated by a population of small cells with round nuclei and scanty cytoplasm. In severely affected regions, hepatocytes were disrupted (FIG. 11C , small windows). These results showed that the anti-β-catenin D-RNAi agent was very effective in knocking out the targeted gene expression at a very low dose of 25 nM and was effective over a long period of time (>10 days). Furthermore, the anti-β-catenin D-RNAi gene silencing effect appeared to be very specific, as non-targeted organs appeared to be normal, indicating that the D-RNAi hybrid compositions had no overt toxicity. The gene silencing in chicken and mice by the D-RNAi agent (FIGS. 11 and 12 ) presents a great potential of localized transgene-like approach in creating animal models for human diseases. - To test in an adult animal model (
FIG. 12 ), patched albino (white) skins of melanin-knockout mice (Rosa-26 strain) were created by a succession of intra-cutaneous (i.c.) transduction of about 50 nM anti-tyrosinase (tyr) mRNA-cDNA hybrids for 4 days (a total of 200 nM). Tyr, a type-I membrane protein and copper-containing enzyme, catalyzes the critical and rate-limiting step of tyrosine hydroxylation in the biosynthesis of melanin (black pigment) in skins and hairs. After 14-day incubation, the expression of melanin was blocked due to the loss of its intermediates resulted from the tyr silencing effect. In contrast, the blank control and dsRNA-transfected mice showed normal skin color (black), indicating that the loss of melanin is specifically caused by the mRNA-cDNA transfection. Moreover, Northern blot analysis using RNA-PCR-derived mRNAs from hair follicles showed a 76.1±5.3% reduction intyr expression 2 days after the transfection of the D-RNAi agent, which was consistent with the immunohistochemistry results from the same skin area, whereas mild, non-specific degradation of common gene transcripts was detected in the dsRNA-transfected skins, shown by the smearing patterns of both the house-keeping control GAPDH and tyr mRNAs in Northern blots (4th column, left-bottom insert windows). These results show that the utilization of D-RNAi agents provides a powerful new strategy for in vivo gene therapy, potentially to melanoma. At the same dosage (200 nM in total), the D-RNAi transfections did not cause detectable cytotoxicity, while the dsRNA transfections induced noted non-specific mRNA degradation. This even underscores the fact that the mRNA-cDNA hybrids are effective even under in vivo systems without the side-effects of dsRNA. The results also indicate that this gene silencing effect is stable and efficient in knocking out target gene expression over a relatively long period of time since the hair regrowth requires at least a ten-day recovery. Further, it was observed that non-targeted skin hairs appeared to be normal, indicating that the compositions used herein possess high specificity and no overt toxicity. Thus, the D-RNAi-based gene manipulation offers the advantages of low in vivo dosage, stability, long-term effectiveness, and lack of overt toxicity. - A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
Claims (2)
1. An isolated RNA comprising an intron RNA that is released in a cell, thereby modulating the function of a target gene, wherein the isolated RNA does not contain a combination of a splice donor site that includes 5′-GU(A/G)AGU-3′ and a splice acceptor site that includes 5′-CU(A/G)A(C/U)NG-3′.
2-57. (canceled)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/084,512 US20150031871A1 (en) | 2002-09-16 | 2013-11-19 | RNA-Mediated Gene Modulation |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US41106202P | 2002-09-16 | 2002-09-16 | |
US41840502P | 2002-10-12 | 2002-10-12 | |
US10/663,875 US8609831B2 (en) | 2002-09-16 | 2003-09-16 | RNA-mediated gene modulation |
US14/084,512 US20150031871A1 (en) | 2002-09-16 | 2013-11-19 | RNA-Mediated Gene Modulation |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/663,875 Division US8609831B2 (en) | 2002-09-16 | 2003-09-16 | RNA-mediated gene modulation |
Publications (1)
Publication Number | Publication Date |
---|---|
US20150031871A1 true US20150031871A1 (en) | 2015-01-29 |
Family
ID=31998011
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/663,875 Expired - Fee Related US8609831B2 (en) | 2002-09-16 | 2003-09-16 | RNA-mediated gene modulation |
US14/084,512 Abandoned US20150031871A1 (en) | 2002-09-16 | 2013-11-19 | RNA-Mediated Gene Modulation |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/663,875 Expired - Fee Related US8609831B2 (en) | 2002-09-16 | 2003-09-16 | RNA-mediated gene modulation |
Country Status (3)
Country | Link |
---|---|
US (2) | US8609831B2 (en) |
AU (1) | AU2003270734A1 (en) |
WO (1) | WO2004024940A2 (en) |
Families Citing this family (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6878269B2 (en) * | 1996-01-31 | 2005-04-12 | Kaneka Corporation | Device for body fluid purification and system for body fluid purification |
GB9925459D0 (en) * | 1999-10-27 | 1999-12-29 | Plant Bioscience Ltd | Gene silencing |
US20060257851A1 (en) * | 2002-11-26 | 2006-11-16 | Itzhak Bentwich | Bioinformatically detectable group of novel viral regulatory genes and uses thereof |
US9453219B2 (en) * | 2003-05-15 | 2016-09-27 | Mello Biotech Taiwan Co., Ltd. | Cosmetic designs and products using intronic RNA |
AU2005243410B2 (en) | 2004-05-14 | 2010-04-22 | Rosetta Genomics Ltd. | Micronas and uses thereof |
US7795419B2 (en) * | 2004-05-26 | 2010-09-14 | Rosetta Genomics Ltd. | Viral and viral associated miRNAs and uses thereof |
US20060200878A1 (en) | 2004-12-21 | 2006-09-07 | Linda Lutfiyya | Recombinant DNA constructs and methods for controlling gene expression |
WO2006073727A2 (en) | 2004-12-21 | 2006-07-13 | Monsanto Technology, Llc | Recombinant dna constructs and methods for controlling gene expression |
EP3378953A1 (en) | 2006-10-12 | 2018-09-26 | Monsanto Technology LLC | Plant micrornas and methods of use thereof |
KR101532442B1 (en) * | 2007-12-10 | 2015-06-29 | 고쿠리츠 다이가쿠 호진 교토 다이가쿠 | Efficient method for nuclear reprogramming |
JP5626619B2 (en) * | 2008-12-08 | 2014-11-19 | 国立大学法人京都大学 | Efficient nuclear initialization method |
WO2009079606A2 (en) * | 2007-12-17 | 2009-06-25 | University Of Southern California | Microrna-induced es-like cells and uses thereof |
WO2009091659A2 (en) * | 2008-01-16 | 2009-07-23 | Shi-Lung Lin | Generation of tumor-free embryonic stem-like pluripotent cells using inducible recombinant rna agents |
CN103189511B (en) | 2010-07-12 | 2016-10-12 | 国立大学法人鸟取大学 | Utilize the novel hiPSC facture that siRNA imports |
US20150307894A1 (en) | 2012-11-28 | 2015-10-29 | Monsanto Technology Llc | Transgenic Plants With Enhanced Traits |
WO2018098352A2 (en) | 2016-11-22 | 2018-05-31 | Jun Oishi | Targeting kras induced immune checkpoint expression |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6710174B2 (en) * | 2001-09-13 | 2004-03-23 | Isis Pharmaceuticals, Inc. | Antisense inhibition of vascular endothelial growth factor receptor-1 expression |
JP4321877B2 (en) * | 1995-12-15 | 2009-08-26 | バークシス コーポレイション | Therapeutic molecules produced by trans-splicing |
US6506559B1 (en) | 1997-12-23 | 2003-01-14 | Carnegie Institute Of Washington | Genetic inhibition by double-stranded RNA |
EP2210948A3 (en) * | 1999-12-10 | 2010-10-06 | Life Technologies Corporation | Use of multiple recombination sites with unique specificity in recombinational cloning |
-
2003
- 2003-09-16 US US10/663,875 patent/US8609831B2/en not_active Expired - Fee Related
- 2003-09-16 WO PCT/US2003/029274 patent/WO2004024940A2/en not_active Application Discontinuation
- 2003-09-16 AU AU2003270734A patent/AU2003270734A1/en not_active Abandoned
-
2013
- 2013-11-19 US US14/084,512 patent/US20150031871A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
WO2004024940A3 (en) | 2004-08-19 |
US20040253604A1 (en) | 2004-12-16 |
WO2004024940A2 (en) | 2004-03-25 |
US8609831B2 (en) | 2013-12-17 |
AU2003270734A1 (en) | 2004-04-30 |
AU2003270734A8 (en) | 2004-04-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20150031871A1 (en) | RNA-Mediated Gene Modulation | |
US5712384A (en) | Ribozymes targeting retroviral packaging sequence expression constructs and recombinant retroviruses containing such constructs | |
US20060228800A1 (en) | Novel Transgenic Methods Using intronic RNA | |
CA2055435A1 (en) | Stably transformed eucaryotic cells comprising a foreign transcribable dna under the control of a pol iii promoter | |
JP2010537639A (en) | Asymmetric interfering RNA compositions and uses thereof | |
JP2746480B2 (en) | Vectors with multiple target response factors affecting gene expression | |
US20060079471A1 (en) | Snornai-small nucleolar rna degradation by rna interference in trypanosomatids | |
US20040106566A1 (en) | RNA-splicing and processing-directed gene silencing and the relative applications thereof | |
US20120301449A1 (en) | Rna interference target for treating aids | |
AU698730B2 (en) | Ribozymes targeting the retroviral packaging sequence expression construct s and recombinant retroviruses containing such constructs | |
JPH06506599A (en) | Inhibition of retroviruses by antisense nucleic acids complementary to packaging sequences | |
US8299043B2 (en) | Treating glaucoma, cardiovascular diseases, and renal diseases | |
US20060269530A1 (en) | RNA interference compositions and methods | |
AU703964B2 (en) | Ribozymes targeting the retroviral packaging sequence expression constructs and recombinant retroviruses containing such constructs | |
Cagnon et al. | Protection of a T-cell line from human immunodeficiency virus replication by the stable expression of a short antisense RNA sequence carried by a shuttle RNA molecule | |
JP2022518808A (en) | Antisense oligonucleotides for the treatment of Laver congenital amaurosis | |
HEUSCH et al. | Intracellular Immunization against SIVmacUtilizing a Hairpin Ribozyme | |
CN114908089B (en) | Construction method and application of 3' UTR | |
Kim et al. | Examination of antisense RNA and oligodeoxynucleotides as potential inhibitors of avian leukosis virus replication in RP30 cells | |
Ganousis et al. | Improved accumulation and activity of ribozymes expressed from tRNA-based RNA polymerase III promoter | |
AU2008251037A1 (en) | Suppression of viruses involved in respiratory infection or disease | |
SYNTHESIZED | INHIBITION OF INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS REPLICATION | |
EP1111057A1 (en) | Prodrug ribozyme | |
Whyte | Cationic liposome-mediated transfection of mammary epithelial cells with AAV-based plasmid DNA | |
Wongwit | Structural and physiological changes observed in schistosomula of Schistosoma mansoni as the effects of antisense oligodeoxynucleotides |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF SOUTHERN CALIFORNIA;REEL/FRAME:035269/0766 Effective date: 20150319 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: NATIONAL INSTITUTES OF HEALTH, MARYLAND Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF SOUTHERN CALIFORNIA;REEL/FRAME:068053/0786 Effective date: 20240722 |