JP7413253B2 - Casヌクレアーゼの特異的活性化のための抗CRISPRポリペプチドの使用 - Google Patents
Casヌクレアーゼの特異的活性化のための抗CRISPRポリペプチドの使用 Download PDFInfo
- Publication number
- JP7413253B2 JP7413253B2 JP2020508574A JP2020508574A JP7413253B2 JP 7413253 B2 JP7413253 B2 JP 7413253B2 JP 2020508574 A JP2020508574 A JP 2020508574A JP 2020508574 A JP2020508574 A JP 2020508574A JP 7413253 B2 JP7413253 B2 JP 7413253B2
- Authority
- JP
- Japan
- Prior art keywords
- polynucleotide
- sequence
- polypeptide
- acr
- host cell
- 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.)
- Active
Links
- 101710163270 Nuclease Proteins 0.000 title claims description 90
- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 89
- 229920001184 polypeptide Polymers 0.000 title claims description 87
- 102000004196 processed proteins & peptides Human genes 0.000 title claims description 87
- 238000010354 CRISPR gene editing Methods 0.000 title claims description 28
- 230000004913 activation Effects 0.000 title description 16
- 102000040430 polynucleotide Human genes 0.000 claims description 228
- 108091033319 polynucleotide Proteins 0.000 claims description 228
- 239000002157 polynucleotide Substances 0.000 claims description 228
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 claims description 102
- 230000009368 gene silencing by RNA Effects 0.000 claims description 102
- 239000002679 microRNA Substances 0.000 claims description 91
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 88
- 239000013598 vector Substances 0.000 claims description 72
- 108091033409 CRISPR Proteins 0.000 claims description 67
- 150000007523 nucleic acids Chemical group 0.000 claims description 66
- 230000014509 gene expression Effects 0.000 claims description 50
- 238000000034 method Methods 0.000 claims description 46
- 239000012634 fragment Substances 0.000 claims description 43
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 42
- 108090000623 proteins and genes Proteins 0.000 claims description 39
- 108020005004 Guide RNA Proteins 0.000 claims description 37
- 230000000295 complement effect Effects 0.000 claims description 24
- 108091070501 miRNA Proteins 0.000 claims description 19
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 16
- 239000003814 drug Substances 0.000 claims description 8
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 description 197
- 108700011259 MicroRNAs Proteins 0.000 description 74
- 230000000694 effects Effects 0.000 description 56
- 108091007780 MiR-122 Proteins 0.000 description 54
- 108091051828 miR-122 stem-loop Proteins 0.000 description 54
- 210000001519 tissue Anatomy 0.000 description 42
- 239000004055 small Interfering RNA Substances 0.000 description 36
- 206010028980 Neoplasm Diseases 0.000 description 29
- 108020004459 Small interfering RNA Proteins 0.000 description 29
- 201000011510 cancer Diseases 0.000 description 25
- 230000002452 interceptive effect Effects 0.000 description 24
- 108020004414 DNA Proteins 0.000 description 22
- 239000002773 nucleotide Substances 0.000 description 22
- 125000003729 nucleotide group Chemical group 0.000 description 22
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 21
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 17
- 208000026350 Inborn Genetic disease Diseases 0.000 description 16
- 208000016361 genetic disease Diseases 0.000 description 16
- 230000004770 neurodegeneration Effects 0.000 description 16
- 208000015122 neurodegenerative disease Diseases 0.000 description 16
- 102000039446 nucleic acids Human genes 0.000 description 16
- 108020004707 nucleic acids Proteins 0.000 description 16
- 201000010099 disease Diseases 0.000 description 15
- 239000008194 pharmaceutical composition Substances 0.000 description 15
- 150000001875 compounds Chemical class 0.000 description 14
- 238000001890 transfection Methods 0.000 description 14
- 208000035473 Communicable disease Diseases 0.000 description 13
- 108700019146 Transgenes Proteins 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 13
- 238000011282 treatment Methods 0.000 description 13
- 108090000331 Firefly luciferases Proteins 0.000 description 12
- 108020004999 messenger RNA Proteins 0.000 description 12
- 238000009396 hybridization Methods 0.000 description 11
- 108060001084 Luciferase Proteins 0.000 description 10
- 239000005089 Luciferase Substances 0.000 description 10
- 230000001419 dependent effect Effects 0.000 description 10
- 239000013604 expression vector Substances 0.000 description 10
- 210000004962 mammalian cell Anatomy 0.000 description 10
- 230000008685 targeting Effects 0.000 description 10
- 108020005345 3' Untranslated Regions Proteins 0.000 description 9
- 230000015556 catabolic process Effects 0.000 description 9
- 238000006731 degradation reaction Methods 0.000 description 9
- 230000001404 mediated effect Effects 0.000 description 9
- 230000035772 mutation Effects 0.000 description 9
- 108010042407 Endonucleases Proteins 0.000 description 8
- 102000004533 Endonucleases Human genes 0.000 description 8
- 108010052090 Renilla Luciferases Proteins 0.000 description 8
- 101000910035 Streptococcus pyogenes serotype M1 CRISPR-associated endonuclease Cas9/Csn1 Proteins 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 241000254158 Lampyridae Species 0.000 description 7
- 241000242739 Renilla Species 0.000 description 7
- 108091027967 Small hairpin RNA Proteins 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 239000013641 positive control Substances 0.000 description 7
- 239000013607 AAV vector Substances 0.000 description 6
- 241000702421 Dependoparvovirus Species 0.000 description 6
- 241000193996 Streptococcus pyogenes Species 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 6
- 208000035475 disorder Diseases 0.000 description 6
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 238000013518 transcription Methods 0.000 description 6
- 230000035897 transcription Effects 0.000 description 6
- 239000013603 viral vector Substances 0.000 description 6
- 230000003612 virological effect Effects 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 102000000574 RNA-Induced Silencing Complex Human genes 0.000 description 5
- 108010016790 RNA-Induced Silencing Complex Proteins 0.000 description 5
- 208000009415 Spinocerebellar Ataxias Diseases 0.000 description 5
- 108091023045 Untranslated Region Proteins 0.000 description 5
- 239000000969 carrier Substances 0.000 description 5
- 230000003197 catalytic effect Effects 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 239000003937 drug carrier Substances 0.000 description 5
- 230000009977 dual effect Effects 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 238000012239 gene modification Methods 0.000 description 5
- 230000005017 genetic modification Effects 0.000 description 5
- 235000013617 genetically modified food Nutrition 0.000 description 5
- 239000004615 ingredient Substances 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 230000001225 therapeutic effect Effects 0.000 description 5
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 101150063416 add gene Proteins 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 201000003624 spinocerebellar ataxia type 1 Diseases 0.000 description 4
- 208000024827 Alzheimer disease Diseases 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 3
- 206010013801 Duchenne Muscular Dystrophy Diseases 0.000 description 3
- 208000023105 Huntington disease Diseases 0.000 description 3
- 241000186779 Listeria monocytogenes Species 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 210000004102 animal cell Anatomy 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 230000030279 gene silencing Effects 0.000 description 3
- 238000001415 gene therapy Methods 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 210000003494 hepatocyte Anatomy 0.000 description 3
- -1 host cells Substances 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000012678 infectious agent Substances 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 230000009752 translational inhibition Effects 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 2
- IZHVBANLECCAGF-UHFFFAOYSA-N 2-hydroxy-3-(octadecanoyloxy)propyl octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)COC(=O)CCCCCCCCCCCCCCCCC IZHVBANLECCAGF-UHFFFAOYSA-N 0.000 description 2
- 101150109930 ACR gene Proteins 0.000 description 2
- 101150005393 CBF1 gene Proteins 0.000 description 2
- 101100161882 Caenorhabditis elegans acr-3 gene Proteins 0.000 description 2
- 201000003728 Centronuclear myopathy Diseases 0.000 description 2
- 102100022641 Coagulation factor IX Human genes 0.000 description 2
- 102100026735 Coagulation factor VIII Human genes 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 101100329224 Coprinopsis cinerea (strain Okayama-7 / 130 / ATCC MYA-4618 / FGSC 9003) cpf1 gene Proteins 0.000 description 2
- 201000003883 Cystic fibrosis Diseases 0.000 description 2
- 201000003542 Factor VIII deficiency Diseases 0.000 description 2
- 208000009292 Hemophilia A Diseases 0.000 description 2
- 208000029433 Herpesviridae infectious disease Diseases 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 241000272168 Laridae Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 208000009608 Papillomavirus Infections Diseases 0.000 description 2
- 208000018737 Parkinson disease Diseases 0.000 description 2
- 102000010292 Peptide Elongation Factor 1 Human genes 0.000 description 2
- 108010077524 Peptide Elongation Factor 1 Proteins 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 208000032978 Structural Congenital Myopathies Diseases 0.000 description 2
- 108091028113 Trans-activating crRNA Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 101150059443 cas12a gene Proteins 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 238000012226 gene silencing method Methods 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- 208000007345 glycogen storage disease Diseases 0.000 description 2
- 208000009429 hemophilia B Diseases 0.000 description 2
- 208000002672 hepatitis B Diseases 0.000 description 2
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000017730 intein-mediated protein splicing Effects 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 108091028606 miR-1 stem-loop Proteins 0.000 description 2
- 108091063796 miR-206 stem-loop Proteins 0.000 description 2
- 108091071651 miR-208 stem-loop Proteins 0.000 description 2
- 108091084446 miR-208a stem-loop Proteins 0.000 description 2
- 108091062547 miR-208a-1 stem-loop Proteins 0.000 description 2
- 108091055375 miR-208a-2 stem-loop Proteins 0.000 description 2
- 108091007420 miR‐142 Proteins 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000001788 mono and diglycerides of fatty acids Substances 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000000737 periodic effect Effects 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- FGDZQCVHDSGLHJ-UHFFFAOYSA-M rubidium chloride Chemical compound [Cl-].[Rb+] FGDZQCVHDSGLHJ-UHFFFAOYSA-M 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 208000007056 sickle cell anemia Diseases 0.000 description 2
- 210000002027 skeletal muscle Anatomy 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000010972 statistical evaluation Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 206010001197 Adenocarcinoma of the cervix Diseases 0.000 description 1
- 208000034246 Adenocarcinoma of the cervix uteri Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102100036826 Aldehyde oxidase Human genes 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 208000002267 Anti-neutrophil cytoplasmic antibody-associated vasculitis Diseases 0.000 description 1
- 244000105975 Antidesma platyphyllum Species 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- 102000014461 Ataxins Human genes 0.000 description 1
- 108010078286 Ataxins Proteins 0.000 description 1
- 241000701822 Bovine papillomavirus Species 0.000 description 1
- XMWRBQBLMFGWIX-UHFFFAOYSA-N C60 fullerene Chemical class C12=C3C(C4=C56)=C7C8=C5C5=C9C%10=C6C6=C4C1=C1C4=C6C6=C%10C%10=C9C9=C%11C5=C8C5=C8C7=C3C3=C7C2=C1C1=C2C4=C6C4=C%10C6=C9C9=C%11C5=C5C8=C3C3=C7C1=C1C2=C4C6=C2C9=C5C3=C12 XMWRBQBLMFGWIX-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010008025 Cerebellar ataxia Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 108091028732 Concatemer Proteins 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000589601 Francisella Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 101150094690 GAL1 gene Proteins 0.000 description 1
- 102100028501 Galanin peptides Human genes 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 description 1
- 101100121078 Homo sapiens GAL gene Proteins 0.000 description 1
- 101000780643 Homo sapiens Protein argonaute-2 Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102100025169 Max-binding protein MNT Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 108091030146 MiRBase Proteins 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 108091093105 Nuclear DNA Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 241000701945 Parvoviridae Species 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 102000002508 Peptide Elongation Factors Human genes 0.000 description 1
- 108010068204 Peptide Elongation Factors Proteins 0.000 description 1
- 102100034207 Protein argonaute-2 Human genes 0.000 description 1
- 241001148570 Rhodothermus marinus Species 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 1
- 229940090047 auto-injector Drugs 0.000 description 1
- 201000004562 autosomal dominant cerebellar ataxia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 108091005948 blue fluorescent proteins Proteins 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 201000006662 cervical adenocarcinoma Diseases 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000002074 deregulated effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000010864 dual luciferase reporter gene assay Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000008995 epigenetic change Effects 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- 230000004049 epigenetic modification Effects 0.000 description 1
- 229910003472 fullerene Inorganic materials 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 102000018146 globin Human genes 0.000 description 1
- 108060003196 globin Proteins 0.000 description 1
- 229940074045 glyceryl distearate Drugs 0.000 description 1
- 229940075507 glyceryl monostearate Drugs 0.000 description 1
- 235000009424 haa Nutrition 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000002161 motor neuron Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000030648 nucleus localization Effects 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 102000005912 ran GTP Binding Protein Human genes 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 229940102127 rubidium chloride Drugs 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000009919 sequestration Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000002924 silencing RNA Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 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
- 231100001274 therapeutic index Toxicity 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 108091008023 transcriptional regulators Proteins 0.000 description 1
- 108091006107 transcriptional repressors Proteins 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 108091005957 yellow fluorescent proteins Proteins 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
-
- 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.
- C12N2310/141—MicroRNAs, miRNAs
-
- 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
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicinal Preparation (AREA)
Description
(I)(i)抗CRISPR(Acr)ポリペプチドをコードする配列および(ii)RNA干渉(RNAi)標的配列を含む第1のポリヌクレオチド;ならびにCasヌクレアーゼまたはCasヌクレアーゼをコードする発現可能な遺伝子を含む第2のポリヌクレオチド;
(II)(i)抗CRISPR(Acr)ポリペプチドをコードする配列および(ii)RNA干渉(RNAi)標的配列を含む第1のポリヌクレオチドであって、Casヌクレアーゼの第1の断片をコードする発現可能な遺伝子をさらに含む前記第1のポリヌクレオチド;ならびにCasヌクレアーゼの第2の断片をコードする発現可能な遺伝子を含む第2のポリヌクレオチドであって、前記Casヌクレアーゼの前記第1および第2の断片が活性なCasヌクレアーゼを一緒になって再構成する、第1および第2のポリヌクレオチド;または
(III)(i)抗CRISPR(Acr)ポリペプチドをコードする配列、(ii)RNA干渉(RNAi)標的配列、および(iii)Casヌクレアーゼをコードする配列を含む第1のポリヌクレオチド;ならびに阻害的RNA、好ましくは、前記RNAi標的配列にハイブリダイズするsiRNAもしくはmiRNA、より好ましくは、前記RNAi標的配列にハイブリダイズするsiRNAをコードする発現可能な遺伝子を含む第2のポリヌクレオチド
を含む、キットに関する。
a1)前記宿主細胞を、本発明によるポリヌクレオチドと;本発明によるベクターと;および/または本発明による宿主細胞と接触させるステップ;さらに、前記宿主細胞を、Casヌクレアーゼ活性およびgRNAと接触させるステップ;または
a2)前記宿主細胞を、本発明によるキットに含まれる成分およびgRNAと接触させるステップ;
ならびに
b)それによって、前記宿主細胞を遺伝的に改変するステップ
を含む、方法に関する。
a1)宿主細胞を、本発明によるポリヌクレオチドと;本発明によるベクターと;および/または本発明による宿主細胞と接触させるステップ;さらに、前記宿主細胞を、Casヌクレアーゼ活性およびgRNAと接触させるステップ;または
a2)前記宿主細胞を、本発明によるキットに含まれる成分およびgRNAと接触させるステップ;ならびに
b)それによって、前記対象における遺伝的疾患、神経変性疾患、がん、および/または感染症を処置するステップ
を含む方法に関する。
a)配列番号21の核酸配列;
b)配列番号21の配列と少なくとも70%同一である核酸配列;
c)配列番号20のアミノ酸配列を含むポリペプチドをコードする核酸配列;または
d)配列番号20の配列と少なくとも70%同一であるアミノ酸配列を含むポリペプチドをコードする核酸配列
を含む、実施形態1または2に記載のポリヌクレオチド。
(II)(i)抗CRISPR(Acr)ポリペプチドをコードする配列および(ii)RNA干渉(RNAi)標的配列を含む第1のポリヌクレオチドであって、Casヌクレアーゼの第1の断片をコードする発現可能な遺伝子をさらに含む前記第1のポリヌクレオチド、ならびにCasヌクレアーゼの第2の断片をコードする発現可能な遺伝子を含む第2のポリヌクレオチドであって、前記Casヌクレアーゼの前記第1および第2の断片が、活性なCasヌクレアーゼを一緒になって再構成する、第1および第2のポリヌクレオチド;または
(III)(i)抗CRISPR(Acr)ポリペプチドをコードする配列、(ii)RNA干渉(RNAi)標的配列、および(iii)Casヌクレアーゼをコードする配列を含む第1のポリヌクレオチド;ならびに阻害的RNA、好ましくは、前記RNAi標的配列にハイブリダイズするsiRNA、shRNAもしくはmiRNA、より好ましくは、前記RNA媒介性分解標的配列にハイブリダイズするsiRNAもしくはshRNAをコードする発現可能な遺伝子を含む第2のポリヌクレオチド
を含む、キット。
a1)前記宿主細胞を、実施形態1~16のいずれか1つに記載のポリヌクレオチドと;実施形態17もしくは18に記載のベクターと;および/または実施形態19に記載の宿主細胞と接触させるステップ;さらに、前記宿主細胞を、Casヌクレアーゼ活性およびgRNAと接触させるステップ;または
a2)前記宿主細胞を、実施形態23~30のいずれか1つに記載のキットに含まれる成分と接触させるステップ;
ならびに
b)それによって、前記宿主細胞を遺伝的に改変するステップ
を含む、方法。
a1)宿主細胞を、実施形態1~16のいずれか1つに記載のポリヌクレオチドと;実施形態17もしくは18に記載のベクターと;および/または実施形態19に記載の宿主細胞と接触させるステップ;さらに、前記宿主細胞を、Casヌクレアーゼ活性およびgRNAと接触させるステップ;または
a2)前記宿主細胞を、実施形態23~30のいずれか1つに記載のキットに含まれる成分と接触させるステップ;
ならびに
b)それによって、前記対象における遺伝的疾患、神経変性疾患、がん、および/または感染症を処置するステップ
を含む方法。
(実施例)
以下の実施例は、単に本発明を例示するものである。それらは、何であれ、本発明の範囲を限定すると解釈されるべきではない。
1.1 プラスミド
pAAV-SMVP-Cas9N (Addgene #80930)およびpAAV-SMVP-Cas9C (Addgene #80931)は、George Churchからの贈り物であった。本明細書で使用されるさらなるベクターは、配列番号3~19を有する(Nihongakiら、(2015), Chem Biol. 22(2):169-74; Konermanら(2015), Nature 517(7536):583-8を参照)。
ヒト子宮頸部腺癌細胞(HELA)およびヒト肝癌細胞(Huh-7)を、加湿組織培養インキュベーター中、37℃および5%CO2で維持した。細胞を、75cm2のフラスコ(STARLAB)(表1)中で培養し、2~3日毎に継代した。
実験のために、細胞を透明な、組織培養処理された96ウェルプレート(STARLAB)に播種した(ウェルあたり6,000個の細胞および100μlの培地中)。トランスフェクションを、播種の12~16時間後に行った。Lipofectamine(商標)3000(Invitrogen)を、製造業者のプロトコールに従って全てのトランスフェクションについて使用した。
トランスフェクションの48時間後、ホタルおよびRenillaルシフェラーゼ活性を、製造業者の推奨に従ってDual Luciferase(登録商標)Reporter Assay System (Promega)を用いて決定した。簡単に述べると、培地を吸引し、細胞を100μlのPBSで洗浄した。30μlの供給された溶解バッファーを各ウェルに添加し、室温、450rpmで30分間、プレートを振とうした。次いで、10μlの溶解物を白色の96ウェルプレート(greiner bio-one)に移し、自動注入器を装備したGloMax(登録商標)96 Microplate Luminometer (Promega) を用いて発光を測定した。第1に、35μlのルシフェラーゼアッセイ試薬IIを添加することによって、ホタルルシフェラーゼシグナルを測定した。第2に、反応をクエンチし、35μlのStop & Glo(登録商標)試薬を添加することによって、Renillaルシフェラーゼ反応を同時に開始させた。シグナル統合時間は、10s、次いで、2sの遅延であった。データ分析のために、ホタルの値を、Renillaの値で除算した。データは、平均±3回の実験反復物の平均の標準誤差である。
本発明者らは、可変的AcrIIA4駆動性プロモーター(サイトメガロウイルスプロモーター、CMV;伸長因子アルファプロモーター、EF-1α;EF-1α短縮プロモーター、EFS)を有するモジュラーAcrIIA4発現構築物ならびに任意のmiRNA標的部位の単純な挿入のためのモジュラー3'UTRのセットを生成した(図1A)。次いで、本発明者らは、miR-122のための2つの標的部位のコンカテマーを、本発明者らのAcrIIA4構築物に導入し、miRNAは、任意の他の細胞型ではなく、肝細胞中で特異的に発現された(図1B)。
本発明は、以下の態様を包含する。
[1](i)抗CRISPR(Acr)ポリペプチドをコードする配列及び(ii)miRNA標的配列を含むポリヌクレオチド。
[2]前記miRNAが組織特異的及び/又は細胞型特異的miRNAである、[1]記載のポリヌクレオチド。
[3]同じmiRNAと本質的に相補的な少なくとも2つのmiRNA標的配列、より好ましくは、miRNAの同じ核酸配列と本質的に相補的である少なくとも2つの標的配列を含む、[1]又は[2]記載のポリヌクレオチド。
[4]少なくとも2つの非同一のmiRNA標的配列、より好ましくは、互いに80%未満、さらにより好ましくは、70%未満、最も好ましくは、60%未満同一である少なくとも2つのmiRNA標的配列を含む、[1]~[3]のいずれか1項記載のポリヌクレオチド。
[5]Acrポリペプチドをコードする前記配列が、前記miRNA標的配列、好ましくは、前記発現可能な核酸配列に含まれる全てのmiRNA標的配列の上流に位置する、[1]~[4]のいずれか1項記載のポリヌクレオチド。
[6]前記AcrポリペプチドがAcrIIA4ポリペプチドである、[1]~[5]のいずれか1項記載のポリヌクレオチド。
[7]Acrポリペプチドをコードする前記配列が、
a)配列番号21の核酸配列;
b)配列番号21の配列と少なくとも70%同一である核酸配列;
c)配列番号20のアミノ酸配列を含むポリペプチドをコードする核酸配列;又は
d)配列番号20の配列と少なくとも70%同一であるアミノ酸配列を含むポリペプチドをコードする核酸配列
を含む、[1]~[6]のいずれか1項記載のポリヌクレオチド。
[8]CRISPR関連(Cas)ヌクレアーゼの少なくとも断片をコードする核酸配列をさらに含む、[1]~[7]のいずれか1項記載のポリヌクレオチド。
[9][1]~[8]のいずれか1項記載のポリヌクレオチドを含むベクター。
[10][1]~[8]のいずれか1項記載のポリヌクレオチド及び/又は[9]記載のベクターを含む宿主細胞。
[11]医学における使用のため、好ましくは、遺伝的疾患、神経変性疾患、がん、及び/又は感染症の処置における使用のための、[1]~[8]のいずれか1項記載のポリヌクレオチド及び/又は[9]記載のベクター及び/又は[10]記載の宿主細胞。
[12](I)(i)抗CRISPR(Acr)ポリペプチドをコードする配列及び(ii)RNA干渉(RNAi)標的配列を含む第1のポリヌクレオチド;並びにCasヌクレアーゼ、又はCasヌクレアーゼをコードする発現可能な遺伝子を含む第2のポリヌクレオチド;
(II)(i)抗CRISPR(Acr)ポリペプチドをコードする配列及び(ii)RNA干渉(RNAi)標的配列を含む第1のポリヌクレオチドであって、Casヌクレアーゼの第1の断片をコードする発現可能な遺伝子をさらに含む前記第1のポリヌクレオチド、並びにCasヌクレアーゼの第2の断片をコードする発現可能な遺伝子を含む第2のポリヌクレオチドであって、前記Casヌクレアーゼの前記第1及び第2の断片は、一緒になって活性なCasヌクレアーゼを再構成する、前記第1のポリヌクレオチド及び第2のポリヌクレオチド;又は
(III)(i)抗CRISPR(Acr)ポリペプチドをコードする配列、(ii)RNA干渉(RNAi)標的配列、及び(iii)Casヌクレアーゼをコードする配列を含む第1のポリヌクレオチド;並びに阻害的RNA、好ましくは、前記RNAi標的配列にハイブリダイズするsiRNA若しくはshRNA若しくはmiRNA、より好ましくは、前記RNAi標的配列にハイブリダイズするsiRNAをコードする発現可能な遺伝子を含む第2のポリヌクレオチド
を含む、キット。
[13]前記キットが、少なくとも1つのガイドRNA(gRNA)をコードするポリヌクレオチドを含み、好ましくは、少なくとも1つのgRNAをコードする前記ポリヌクレオチドが、第1又は第2のポリヌクレオチドであり、より好ましくは、第2のポリヌクレオチドである、[12]記載のキット。
[14]宿主細胞を遺伝的に改変するための方法であって、
a1)前記宿主細胞を、[1]~[8]のいずれか1項記載のポリヌクレオチドと;[9]記載のベクターと;及び/又は[10]記載の宿主細胞と接触させ;且つ、さらに、前記宿主細胞を、Casヌクレアーゼ活性及びgRNAと接触させること;又は
a2)前記宿主細胞を、[12]又は[13]記載のキットに含まれる成分と接触させること;
並びに
b)それによって、前記宿主細胞を遺伝的に改変すること
を含む、前記方法。
[15]宿主細胞を遺伝的に改変するための、[1]~[8]のいずれか1項記載のポリヌクレオチド;[9]記載のベクター;[10]記載の宿主細胞;及び/又は[12]若しくは[13]記載のキットの使用。
Claims (15)
- (i)抗CRISPR(Acr)ポリペプチドをコードする配列及び(ii)miRNA標的配列を含むポリヌクレオチド。
- 前記miRNAが組織特異的及び/又は細胞型特異的miRNAである、請求項1記載のポリヌクレオチド。
- 同じmiRNAと相補的な少なくとも2つのmiRNA標的配列を含む、請求項1又は2記載のポリヌクレオチド。
- 少なくとも2つの非同一のmiRNA標的配列を含む、請求項1~3のいずれか1項記載のポリヌクレオチド。
- Acrポリペプチドをコードする前記配列が、前記miRNA標的配列の上流に位置する、請求項1~4のいずれか1項記載のポリヌクレオチド。
- 前記AcrポリペプチドがAcrIIA4ポリペプチドである、請求項1~5のいずれか1項記載のポリヌクレオチド。
- Acrポリペプチドをコードする前記配列が、
a)配列番号21の核酸配列;
b)配列番号21の配列と少なくとも90%同一である核酸配列;
c)配列番号20のアミノ酸配列を含むポリペプチドをコードする核酸配列;又は
d)配列番号20の配列と少なくとも90%同一であるアミノ酸配列を含むポリペプチドをコードする核酸配列
を含む、請求項1~6のいずれか1項記載のポリヌクレオチド。 - CRISPR関連(Cas)ヌクレアーゼの少なくとも断片をコードする核酸配列をさらに含む、請求項1~7のいずれか1項記載のポリヌクレオチド。
- 請求項1~8のいずれか1項記載のポリヌクレオチドを含むベクター。
- 請求項1~8のいずれか1項記載のポリヌクレオチド及び/又は請求項9記載のベクターを含む宿主細胞。
- 医学における使用のための、請求項1~8のいずれか1項記載のポリヌクレオチド及び/又は請求項9記載のベクター及び/又は請求項10記載の宿主細胞。
- (I)(i)抗CRISPR(Acr)ポリペプチドをコードする配列及び(ii)RNA干渉(RNAi)標的配列を含む第1のポリヌクレオチド;並びにCasヌクレアーゼ、又はCasヌクレアーゼをコードする発現可能な遺伝子を含む第2のポリヌクレオチド;
(II)(i)抗CRISPR(Acr)ポリペプチドをコードする配列及び(ii)RNA干渉(RNAi)標的配列を含む第1のポリヌクレオチドであって、Casヌクレアーゼの第1の断片をコードする発現可能な遺伝子をさらに含む前記第1のポリヌクレオチド、並びにCasヌクレアーゼの第2の断片をコードする発現可能な遺伝子を含む第2のポリヌクレオチドであって、前記Casヌクレアーゼの前記第1及び第2の断片は、一緒になって活性なCasヌクレアーゼを再構成する、前記第1のポリヌクレオチド及び第2のポリヌクレオチド;又は
(III)(i)抗CRISPR(Acr)ポリペプチドをコードする配列、(ii)RNA干渉(RNAi)標的配列、及び(iii)Casヌクレアーゼをコードする配列を含む第1のポリヌクレオチド;並びに阻害的RNAをコードする発現可能な遺伝子を含む第2のポリヌクレオチド
を含む、キット。 - 前記キットが、少なくとも1つのガイドRNA(gRNA)をコードするポリヌクレオチドを含む、請求項12記載のキット。
- 宿主細胞を遺伝的に改変するための方法であって、
a1)前記宿主細胞を、請求項1~8のいずれか1項記載のポリヌクレオチド;及び/又は請求項9記載のベクターと接触させ;且つ、さらに、前記宿主細胞を、Casヌクレアーゼ及びgRNAと接触させること;又は
a2)前記宿主細胞を、請求項12又は13記載のキットに含まれるすべての成分と接触させること;
並びに
b)それによって、前記宿主細胞を遺伝的に改変すること
を含む、前記方法。 - 宿主細胞を遺伝的に改変するための、請求項1~8のいずれか1項記載のポリヌクレオチド;請求項9記載のベクター;及び/又は請求項12若しくは13記載のキットの使用。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP17186885.4 | 2017-08-18 | ||
EP17186885.4A EP3444347A1 (en) | 2017-08-18 | 2017-08-18 | Use of anti-crispr polypeptides for specific activation of cas nucleases |
PCT/EP2018/072350 WO2019034784A1 (en) | 2017-08-18 | 2018-08-17 | USE OF ANTI-CRISPRANT POLYPEPTIDES FOR THE SPECIFIC ACTIVATION OF NUCLEASES CASES |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2020532962A JP2020532962A (ja) | 2020-11-19 |
JP7413253B2 true JP7413253B2 (ja) | 2024-01-15 |
Family
ID=59686774
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2020508574A Active JP7413253B2 (ja) | 2017-08-18 | 2018-08-17 | Casヌクレアーゼの特異的活性化のための抗CRISPRポリペプチドの使用 |
Country Status (6)
Country | Link |
---|---|
US (1) | US20200270603A1 (ja) |
EP (2) | EP3444347A1 (ja) |
JP (1) | JP7413253B2 (ja) |
CN (1) | CN111278975A (ja) |
CA (1) | CA3070766A1 (ja) |
WO (1) | WO2019034784A1 (ja) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP7210029B2 (ja) | 2016-11-16 | 2023-01-23 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | CRISPR-Cas9の阻害因子 |
JPWO2020059708A1 (ja) * | 2018-09-17 | 2021-09-02 | 国立大学法人 東京医科歯科大学 | Casタンパク質の活性調節法 |
JP2022112522A (ja) * | 2019-05-31 | 2022-08-03 | 国立大学法人京都大学 | 標的遺伝子を編集する蛋白質を細胞特異的に制御する方法 |
EP3766968A1 (en) * | 2019-07-16 | 2021-01-20 | Deutsches Krebsforschungszentrum | Improving cas nuclease target specificity |
WO2021108442A2 (en) * | 2019-11-27 | 2021-06-03 | The Regents Of The University Of California | Modulators of cas9 polypeptide activity and methods of use thereof |
WO2021158658A1 (en) * | 2020-02-04 | 2021-08-12 | The Board Of Trustees Of The Leland Stanford Junior University | Split crispr-cas for biological signal integration |
CN116355062B (zh) * | 2021-03-04 | 2024-03-26 | 西部(重庆)科学城种质创制大科学中心 | 抗CRISPR蛋白AcrIIIA2SAP26及其应用 |
CN116514935B (zh) * | 2021-03-04 | 2024-04-30 | 西部(重庆)科学城种质创制大科学中心 | RNA编辑系统的抑制剂AcrIIIA1IPLA35及其应用 |
CN113278645B (zh) * | 2021-04-15 | 2022-06-24 | 浙江大学 | 一种增强链霉菌基因组编辑效率的方法及其应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008509705A (ja) | 2004-08-16 | 2008-04-03 | イミューン ディズィーズ インスティテュート インコーポレイテッド | Rna干渉を送達する方法およびその使用法 |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6506559B1 (en) | 1997-12-23 | 2003-01-14 | Carnegie Institute Of Washington | Genetic inhibition by double-stranded RNA |
EP1235842A4 (en) | 1999-10-15 | 2003-04-23 | Univ Massachusetts | GENESIS OF THE RNA INTERFERENCE PATH AS AID OF TARGETED GENTIAN INTERFERENCE |
ES2564823T3 (es) | 2005-05-27 | 2016-03-29 | Ospedale San Raffaele S.R.L. | Vector génico que comprende miARN |
GB201007454D0 (en) * | 2010-05-04 | 2010-06-16 | King S College London | Method of determining tolerance |
WO2012143401A1 (en) * | 2011-04-18 | 2012-10-26 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Tissue-specific regulation of transgene expression |
US10736928B2 (en) * | 2016-01-20 | 2020-08-11 | Richard Brian Murphy, JR. | Pegylated recombinant bacteriophage |
JP7210029B2 (ja) * | 2016-11-16 | 2023-01-23 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | CRISPR-Cas9の阻害因子 |
CN108795989A (zh) * | 2017-04-26 | 2018-11-13 | 哈尔滨工业大学 | SpyCas9的基因编辑活性抑制位点及其抑制剂 |
DE212020000516U1 (de) * | 2019-03-07 | 2022-01-17 | The Regents of the University of California | CRISPR-CAS-Effektorpolypeptide |
EP3766968A1 (en) * | 2019-07-16 | 2021-01-20 | Deutsches Krebsforschungszentrum | Improving cas nuclease target specificity |
-
2017
- 2017-08-18 EP EP17186885.4A patent/EP3444347A1/en not_active Withdrawn
-
2018
- 2018-08-17 EP EP18753201.5A patent/EP3668982A1/en active Pending
- 2018-08-17 CN CN201880054186.5A patent/CN111278975A/zh active Pending
- 2018-08-17 JP JP2020508574A patent/JP7413253B2/ja active Active
- 2018-08-17 CA CA3070766A patent/CA3070766A1/en active Pending
- 2018-08-17 WO PCT/EP2018/072350 patent/WO2019034784A1/en unknown
- 2018-08-17 US US16/639,719 patent/US20200270603A1/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008509705A (ja) | 2004-08-16 | 2008-04-03 | イミューン ディズィーズ インスティテュート インコーポレイテッド | Rna干渉を送達する方法およびその使用法 |
Non-Patent Citations (3)
Title |
---|
Nucleic Acids Research,2015年,Vol. 43,pp. 6450-6458,https://doi.org/10.1093%2Fnar%2Fgkv601 |
Nucleic Acids Research,2017年05月09日,Vol. 45,pp. e118 (1-11),https://doi.org/10.1093/nar/gkx309 |
Science Advances,2017年07月12日,Vol. 3,pp. e1701620 (1-9),https://doi.org/10.1126/sciadv.1701620 |
Also Published As
Publication number | Publication date |
---|---|
CN111278975A (zh) | 2020-06-12 |
WO2019034784A1 (en) | 2019-02-21 |
EP3444347A1 (en) | 2019-02-20 |
EP3668982A1 (en) | 2020-06-24 |
US20200270603A1 (en) | 2020-08-27 |
JP2020532962A (ja) | 2020-11-19 |
CA3070766A1 (en) | 2019-02-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7413253B2 (ja) | Casヌクレアーゼの特異的活性化のための抗CRISPRポリペプチドの使用 | |
JP7094323B2 (ja) | 最適化機能CRISPR-Cas系による配列操作のための系、方法および組成物 | |
US20240117382A1 (en) | DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF CRISPR SYSTEMS AND COMPOSITIONS FOR GENOME EDITING AS TO HEMATOPOIETIC STEM CELLS (HSCs) | |
US11149259B2 (en) | CRISPR-Cas systems and methods for altering expression of gene products, structural information and inducible modular Cas enzymes | |
US20210139872A1 (en) | Crispr having or associated with destabilization domains | |
US20210277371A1 (en) | Engineering of systems, methods and optimized guide compositions with new architectures for sequence manipulation | |
US20240093193A1 (en) | Dead guides for crispr transcription factors | |
US11459557B2 (en) | Use and production of CHD8+/− transgenic animals with behavioral phenotypes characteristic of autism spectrum disorder | |
WO2018005873A1 (en) | Crispr-cas systems having destabilization domain | |
WO2015089351A1 (en) | Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders | |
WO2016049258A2 (en) | Functional screening with optimized functional crispr-cas systems | |
WO2019236081A1 (en) | Targeted gene activation using modified guide rna | |
US20220364122A1 (en) | Bacterial platform for delivery of gene-editing systems to eukaryotic cells | |
Rovai | Developing and improving genome editing as a therapeutic tool for hereditary hemochromatosis | |
Badertscher et al. | Utilizing CRISPR/Cas9 Technologies for in vivo Disease Modeling and Therapy | |
CN117980482A (zh) | Rbm20突变的基因组编辑 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
RD01 | Notification of change of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7426 Effective date: 20210512 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20210512 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20210812 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20220830 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20221130 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230221 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230517 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230718 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20231011 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20231128 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20231227 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 7413253 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |