WO2004056964A2 - Vecteurs pour l'interference arn inductible - Google Patents

Vecteurs pour l'interference arn inductible Download PDF

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Publication number
WO2004056964A2
WO2004056964A2 PCT/US2003/040548 US0340548W WO2004056964A2 WO 2004056964 A2 WO2004056964 A2 WO 2004056964A2 US 0340548 W US0340548 W US 0340548W WO 2004056964 A2 WO2004056964 A2 WO 2004056964A2
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WIPO (PCT)
Prior art keywords
sequence
promoter
nucleic acid
acid construct
label
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PCT/US2003/040548
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English (en)
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WO2004056964A3 (fr
Inventor
William Winston
Ronan C. O'hagan
Joerg Heyer
William Rideout
Bijan Etemad-Moghadam
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Genpath Pharmaceuticals, Incorporated
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Application filed by Genpath Pharmaceuticals, Incorporated filed Critical Genpath Pharmaceuticals, Incorporated
Priority to AU2003299732A priority Critical patent/AU2003299732A1/en
Publication of WO2004056964A2 publication Critical patent/WO2004056964A2/fr
Publication of WO2004056964A3 publication Critical patent/WO2004056964A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised

Definitions

  • Fig. 4B is a bar graph showing that modified human U6 promoters containing a TetO are repressed by TetR.
  • Fig. 5B is a line graph showing that RNAi of luciferase in xenograft tumors expressing (1) luciferase, (2) a luciferase shRNA from a modified human U6 promoter TetO, and (3) a codon optimized TetR can be regulated by doxycycline.
  • Fig. 1 illustrates one vector system of this invention (see Working Examples).
  • a Tet operator sequence (TetOp) is inserted into the promoter region of the vector.
  • TetOp is preferably inserted between the PSE and the transcription initiation site, upstream or downstream from the TATA box. hi some embodiments, the TetOp is immediately adjacent to the TATA box.
  • the expression of the RNAi molecule is thus under the control of tetracycline (or doxycycline, or any other tetracycline analogue). Addition of tetracycline relieves repression of the promoter by a tetracycline repressor that the host cells are also engineered to express.
  • constructs C-E when co-transfected with pCMV Lad, exhibited stronger (approximately 15% more) repression by Lad in the absence of IPTG (i.e., thus less expression of the FFl transcript and less inhibition of luciferase expression), compared to U6 constructs containing only one LacO sequence (e.g., constructs A- B).
  • Fig. 2C shows that constructs A-E inhibited luciferase expression significantly in the absence of Lad expression. The extents of the inhibition among the constructs were comparable. Constructs A and B had a combined average inhibition of 91%, and constructs C-E had a combined average inhibition of 83%.
  • the approximately 400 base pair LoxP Stop cassette consisted of two LoxP sites in the same orientation bracketing six RNA Polymerase III transcriptional termination sites (stretches of four or more Ts from a luciferase gene fragment and a U6 RNA transcriptional termination fragment).
  • the LoxP Stop cassette prevented transcription from proceeding through to the shRNA- coding sequence. Cre-mediated recombination would remove the intervening Stop sequence between the two LoxP sites, leaving only one LoxP site. Transcription could then proceed through to the shRNA-coding sequence.
  • the gpTetR sequence is as follows.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des vecteurs de recombinaison pour l'expression par induction de molécules d'ARN bicaténaire qui interfèrent avec l'expression d'un gène cible.
PCT/US2003/040548 2002-12-18 2003-12-18 Vecteurs pour l'interference arn inductible WO2004056964A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003299732A AU2003299732A1 (en) 2002-12-18 2003-12-18 Vectors for inducible rna interference

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US43485602P 2002-12-18 2002-12-18
US60/434,856 2002-12-18
US49931303P 2003-08-28 2003-08-28
US60/499,313 2003-08-28

Publications (2)

Publication Number Publication Date
WO2004056964A2 true WO2004056964A2 (fr) 2004-07-08
WO2004056964A3 WO2004056964A3 (fr) 2004-10-14

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2003/040548 WO2004056964A2 (fr) 2002-12-18 2003-12-18 Vecteurs pour l'interference arn inductible

Country Status (2)

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AU (1) AU2003299732A1 (fr)
WO (1) WO2004056964A2 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004065613A2 (fr) * 2003-01-17 2004-08-05 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Constructions geniques permettant l'expression inductible de petites molecules d'arn pour une extinction genique ciblee
WO2006048467A2 (fr) * 2004-11-08 2006-05-11 Genoway Animaux modèles comprenant au moins un transgène et une séquence permettant l'expression contrôlée d'un rna interférant avec ledit transgène
EP1731607A1 (fr) * 2005-06-09 2006-12-13 ARTEMIS Pharmaceuticals GmbH Expression au sein d'un organisme vivant de shRNA et siRNA sous le contrôle d'un gène represseur de tétracycline à codons optimisés
WO2006131543A1 (fr) * 2005-06-09 2006-12-14 Artemis Pharmaceuticals Gmbh Expression d'arnsh et d'arnsi dans un organisme vivant sous controle d'un gene represseur de la tetracycline optimise par un codon
WO2007014363A2 (fr) * 2005-07-27 2007-02-01 Genentech, Inc. Vecteurs et procedes les mettant en oeuvre
WO2007035962A2 (fr) * 2005-09-23 2007-03-29 California Institute Of Technology Methode de blocage de gene
EP2290088A1 (fr) * 2004-12-22 2011-03-02 Genentech, Inc. Méthode de production de protéines multi-membranaires
US8530188B2 (en) 2006-02-03 2013-09-10 Fujifilm Diosynth Biotechnologies (UK) Limited Expression system
CN110540995A (zh) * 2018-05-28 2019-12-06 南京农业大学 一种降低肌球蛋白-5蛋白及其在氰基丙烯酸酯类药物抗性治理中的应用
US11884917B2 (en) * 2016-03-17 2024-01-30 Imba—Institut Für Molekulare Biotechnologie Gmbh Conditional CRISPR sgRNA expression

Citations (4)

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US5917122A (en) * 1992-08-26 1999-06-29 Byrne; Guerard Tetracycline repressor-mediated binary regulation system for control of gene expression in transgenic mice
US20040002077A1 (en) * 2001-11-28 2004-01-01 Center For Advanced Science And Technology Incubation, Ltd. siRNA expression system and method for producing functional gene knock-down cell using the system
US20040005593A1 (en) * 2002-03-06 2004-01-08 Rigel Pharmaceuticals, Inc. Novel method for delivery and intracellular synthesis of siRNA molecules
US20040115815A1 (en) * 2002-07-24 2004-06-17 Immusol, Inc. Single promoter system for making siRNA expression cassettes and expression libraries using a polymerase primer hairpin linker

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5917122A (en) * 1992-08-26 1999-06-29 Byrne; Guerard Tetracycline repressor-mediated binary regulation system for control of gene expression in transgenic mice
US20040002077A1 (en) * 2001-11-28 2004-01-01 Center For Advanced Science And Technology Incubation, Ltd. siRNA expression system and method for producing functional gene knock-down cell using the system
US20040005593A1 (en) * 2002-03-06 2004-01-08 Rigel Pharmaceuticals, Inc. Novel method for delivery and intracellular synthesis of siRNA molecules
US20040115815A1 (en) * 2002-07-24 2004-06-17 Immusol, Inc. Single promoter system for making siRNA expression cassettes and expression libraries using a polymerase primer hairpin linker

Non-Patent Citations (8)

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Title
CZAUDERNA FRANK; ET AL: 'Inducible shRNA expression for application in a prostate cancer mouse model' NUCLEIC ACIDS RESEARCH vol. 31, no. 21, 01 November 2003, pages E127-1 - E127-7, XP002291076 *
ELBASHIR S.M. ET AL: 'Analysis of gene function in somatic mammalian cells using small interfering RNAs' METHODS vol. 26, no. 2, February 2002, pages 199 - 213, XP002251055 *
HANNON G.J.: 'RNA interference' NATURE vol. 418, 11 July 2002, pages 244 - 251, XP002979088 *
HASUWA H. ET AL: 'Small interfering RNA and gene silencing in transgenic mice and rats' FEBS LETTERS vol. 532, 13 November 2002, pages 227 - 230, XP004395375 *
OGUETA ET AL: 'Design and in vitro characterization of a single regulatory module for efficient control of gene expression in both plasmid DNA and a self-inactivating lentiviral vector' MOLECULAR MEDICINE vol. 7, no. 8, August 2001, pages 569 - 579, XP002960124 *
OHKAWA ET AL: 'Control of the functional activity of an antisense RNA by a tetracyclin-responsive derivative of the human U6 snRNA promoter' HUMAN GENE THERAPY vol. 11, no. 4, 01 March 2000, pages 577 - 585, XP000926522 *
SASAKI ET AL: 'A system for conditional RNA interference in the mouse using the lac operator - repressor system' SOCIETY FOR NEUROSCIENCE ABSTRACT VIEWER AND ITINERARY PLANNER vol. 2003, no. ABSTRACT NO. 325.4, 08 November 2003, XP002979518 *
SUI ET AL: 'A DNA vector-based RNAi technology to suppress gene expression in mammalian cells' PNAS vol. 99, no. 8, 16 April 2002, pages 5515 - 5520, XP002964701 *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8470797B2 (en) 2003-01-17 2013-06-25 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Inducible small RNA expression constructs for targeted gene silencing
EP2333063A1 (fr) * 2003-01-17 2011-06-15 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Constructions geniques permettant l'expression inductible de petites molecules d'arn pour une extinction genique ciblee
EP2314687A1 (fr) * 2003-01-17 2011-04-27 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Constructions géniques permettant l'expression inductible de petites molécules d'ARN pour une extinction génique ciblée
WO2004065613A2 (fr) * 2003-01-17 2004-08-05 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Constructions geniques permettant l'expression inductible de petites molecules d'arn pour une extinction genique ciblee
US8198077B2 (en) 2003-01-17 2012-06-12 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Inducible small RNA expression constructs for targeted gene silencing
WO2004065613A3 (fr) * 2003-01-17 2004-10-07 Max Planck Gesellschaft Constructions geniques permettant l'expression inductible de petites molecules d'arn pour une extinction genique ciblee
WO2006048467A3 (fr) * 2004-11-08 2006-07-06 Genoway Animaux modèles comprenant au moins un transgène et une séquence permettant l'expression contrôlée d'un rna interférant avec ledit transgène
WO2006048467A2 (fr) * 2004-11-08 2006-05-11 Genoway Animaux modèles comprenant au moins un transgène et une séquence permettant l'expression contrôlée d'un rna interférant avec ledit transgène
FR2877674A1 (fr) * 2004-11-08 2006-05-12 Genoxay Sa Animaux modeles comprenant au moins un transgene et une sequence permettant l'expression controlee d'un rna interferant avec ledit transgene
EP2290086A3 (fr) * 2004-12-22 2011-10-19 Genentech, Inc. Méthode de production de protéines multi-membranaires
EP2290087A3 (fr) * 2004-12-22 2011-06-15 Genentech, Inc. Méthode de production de protéines multi-membranaires
EP2290088A1 (fr) * 2004-12-22 2011-03-02 Genentech, Inc. Méthode de production de protéines multi-membranaires
US8323902B2 (en) 2004-12-22 2012-12-04 Genentech, Inc. Methods for producing soluble membrane-spanning proteins
EP1731607A1 (fr) * 2005-06-09 2006-12-13 ARTEMIS Pharmaceuticals GmbH Expression au sein d'un organisme vivant de shRNA et siRNA sous le contrôle d'un gène represseur de tétracycline à codons optimisés
WO2006131543A1 (fr) * 2005-06-09 2006-12-14 Artemis Pharmaceuticals Gmbh Expression d'arnsh et d'arnsi dans un organisme vivant sous controle d'un gene represseur de la tetracycline optimise par un codon
WO2007014363A2 (fr) * 2005-07-27 2007-02-01 Genentech, Inc. Vecteurs et procedes les mettant en oeuvre
WO2007014363A3 (fr) * 2005-07-27 2007-06-14 Genentech Inc Vecteurs et procedes les mettant en oeuvre
WO2007035962A3 (fr) * 2005-09-23 2007-05-10 California Inst Of Techn Methode de blocage de gene
WO2007035962A2 (fr) * 2005-09-23 2007-03-29 California Institute Of Technology Methode de blocage de gene
US8530188B2 (en) 2006-02-03 2013-09-10 Fujifilm Diosynth Biotechnologies (UK) Limited Expression system
US9677103B2 (en) 2006-02-03 2017-06-13 Fujifilm Diosynth Biotechnologies Uk Limited Expression system
US11098335B2 (en) 2006-02-03 2021-08-24 Fujifilm Diosynth Biotechnologies Uk Limited Expression system
US11884917B2 (en) * 2016-03-17 2024-01-30 Imba—Institut Für Molekulare Biotechnologie Gmbh Conditional CRISPR sgRNA expression
CN110540995A (zh) * 2018-05-28 2019-12-06 南京农业大学 一种降低肌球蛋白-5蛋白及其在氰基丙烯酸酯类药物抗性治理中的应用
CN110540995B (zh) * 2018-05-28 2023-01-10 南京农业大学 一种降低肌球蛋白-5蛋白及其在氰基丙烯酸酯类药物抗性治理中的应用

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Publication number Publication date
WO2004056964A3 (fr) 2004-10-14
AU2003299732A1 (en) 2004-07-14
AU2003299732A8 (en) 2004-07-14

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