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WO2005111219A1 - Procedes et vecteurs d'expression d'arnic - Google Patents

Procedes et vecteurs d'expression d'arnic Download PDF

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Publication number
WO2005111219A1
WO2005111219A1 PCT/US2004/012010 US2004012010W WO2005111219A1 WO 2005111219 A1 WO2005111219 A1 WO 2005111219A1 US 2004012010 W US2004012010 W US 2004012010W WO 2005111219 A1 WO2005111219 A1 WO 2005111219A1
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WO
WIPO (PCT)
Prior art keywords
rna polymerase
polymerase iii
insert
vector
expression
Prior art date
Application number
PCT/US2004/012010
Other languages
English (en)
Inventor
Ajamete Kaykas
Randall T. Moon
Original Assignee
University Of Washington
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by University Of Washington filed Critical University Of Washington
Priority to PCT/US2004/012010 priority Critical patent/WO2005111219A1/fr
Publication of WO2005111219A1 publication Critical patent/WO2005111219A1/fr

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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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 nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function
    • 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron
    • C12N2830/205—Vector systems having a special element relevant for transcription transcription of more than one cistron bidirectional

Definitions

  • the recognition site is typically within the first and second RNA polymerase III promoters.
  • Exemplary restriction enzymes that cleave outside their recognition site comprise Alwl, Bbsl, Bbvl, BceAl, BciVl, BfuAl, Bmrl, Bpml, BpuEl, Bsal, BseRl, Bsgl, BsmAl, BsmBl, Bs Fl, BspMl, Earl, Ecil, Faul, Fokl, Hgal, Hphl, MboII, Mlyl, Mnll, Plel, Sapl, and SfaNl.
  • the restriction enzyme is BsmBl.
  • the first aspect of the invention provides a plurality of expression vectors, each comprising: (a) a first RNA polymerase III promoter operably associated with a first RNA polymerase III termination signal; (b) a second RNA polymerase III promoter operably associated with a second RNA polymerase III termination signal, wherein the first and second RNA polymerase III promoters are oriented to promote bidirectional transcription of an insert disposed between the first and the second RNA polymerase III termination signals; and (c) an insert disposed between the first and second RNA polymerase III termination signals.
  • the invention provides methods for inhibiting expression of a target gene.
  • the vector may be one that is capable of replicating as an extrachromosomal element such as an artificial chromosome or an Epstein Barr-based virus.
  • An "expression cassette" refers to a linear vector according to the invention comprising the regulatory sequences for expressing siRNA molecules and an insert that can be transcribed to produce siRNA molecules.
  • the vectors of the invention include a first and a second RNA polymerase III promoter.
  • RNA polymerase III promoters, RNA polymerase III termination signals, and the insert are operably associated in such a way that from each strand only the insert and two or three thymidines of an RNA polymerase III termination signal are transcribed.
  • Exemplary restriction enzymes that cleave outside of their recognition site include, but are not limited to, Alwl, Bbsl, Bbvl, BceAl, BciVl, BfuAl, Bmrl, Bpml, BpuEl, Bsal, BseRl, Bsgl, BsmAl, BsmBl, BsmFl, BspMl, Earl, Ecil, Faul, Fokl, Hgal, Hphl, MboII, Mlyl, Mnll, Plel, Sapl, and SfaNl.
  • the recognition sites for restriction enzymes are described, for example, in New England BioLabs Catalog & Technical Reference (2002-03), which publication is incorporated herein by reference.
  • the vectors of the invention may also be used to create transgenic organisms, such as transgenic mice, using methods standard in the art (see, for example, Carmell et al. (2003) Nature Struct. Biol. 10:91-2, incorporated herein by reference).
  • Transgenic animals comprising the vectors of the invention provide useful model systems, for example, for studying gene function and associated pathologies, for screening candidate drugs effective to treat conditions associated with genes that are over-expressed or mis-expressed, and for drug target validation (see, e.g., Aza-Blank et al. (2003) Mol. Cell. 12:627-37; Zheng et al. (2003) Proc. Natl. Acad. Sci. U.S.A.
  • an effective amount or dose of the vectors of the invention is well within the capability of those skilled in the art.
  • the amount actually administered will be dependent upon the individual subject, and will preferably be an optimized amount such that the desired effect is achieved without significant side- effects.
  • bidirectional transcription of the target gene-specific insert in the expression vectors produces siRNA molecules that inhibit the expression of the target gene.
  • inhibit the expression of a target gene refers to the reduction of the level of target gene expression or the elimination of expression of the target gene, compared to the expression of the target gene in a host cell that does not contain the expression vector with the target gene-specific insert.
  • the level of expression of the target gene may be monitored by examining RNA expression and/or protein expression.

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

Abstract

Dans un premier aspect, l'invention concerne des vecteurs d'expression comprenant : (a) un premier promoteur d'ARN polymérase III opérationnellement associé à un premier signal de terminaison d'ARN polymérase III, et (b) un second promoteur d'ARN polymérase III opérationnellement associé à un second signal de terminaison d'ARN polymérase III, le premier et le second promoteur d'ARN polymérase III étant orientés de manière à promouvoir la transcription bidirectionnelle d'un insert disposé entre le premier et le second signal de terminaison d'ARN polymérase III. Dans d'autres aspects, l'invention concerne des procédés d'utilisation de ces vecteurs d'expression afin d'inhiber l'expression de gènes cibles, et de déterminer l'effet d'ARNic sur des procédés biologiques, et afin d'identifier les ARNic qui affectent des procédés biologiques.
PCT/US2004/012010 2004-04-16 2004-04-16 Procedes et vecteurs d'expression d'arnic WO2005111219A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2004/012010 WO2005111219A1 (fr) 2004-04-16 2004-04-16 Procedes et vecteurs d'expression d'arnic

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2004/012010 WO2005111219A1 (fr) 2004-04-16 2004-04-16 Procedes et vecteurs d'expression d'arnic

Publications (1)

Publication Number Publication Date
WO2005111219A1 true WO2005111219A1 (fr) 2005-11-24

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/012010 WO2005111219A1 (fr) 2004-04-16 2004-04-16 Procedes et vecteurs d'expression d'arnic

Country Status (1)

Country Link
WO (1) WO2005111219A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006033756A3 (fr) * 2004-08-23 2007-01-18 Nucleonics Inc Constructions d'expression de promoteurs d'arn polymerase iii multiples
WO2009071722A1 (fr) * 2007-12-07 2009-06-11 Newbiotechnic, S.A. PROCÉDÉS ET TROUSSES POUR LA PRÉPARATION DE GÉNOTHÈQUES D'ARNsi SPÉCIFIQUES D'UN TRANSCRIPTOME PAR TRANSCRIPTION CONVERGENTE

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004009794A2 (fr) * 2002-07-24 2004-01-29 Immusol, Inc. Nouvelles banques genomiques de petits fragments d'arn interferents et procedes de production et d'utilisation de ces banques
WO2004011647A1 (fr) * 2002-07-26 2004-02-05 Chiron Corporation Petites molécules modifiées d'arn d'interférence et leurs procédés d'utilisation
WO2004022777A1 (fr) * 2002-09-04 2004-03-18 Johnson & Johnson Research Pty Ltd Procedes d'utilisation d'adnds dans la mediation d'arn interferents (arni)
WO2004024872A2 (fr) * 2002-09-13 2004-03-25 California Institute Of Technology Methode d'expression de petites molecules d'arn antiviral a l'interieur d'une cellule

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004009794A2 (fr) * 2002-07-24 2004-01-29 Immusol, Inc. Nouvelles banques genomiques de petits fragments d'arn interferents et procedes de production et d'utilisation de ces banques
WO2004011647A1 (fr) * 2002-07-26 2004-02-05 Chiron Corporation Petites molécules modifiées d'arn d'interférence et leurs procédés d'utilisation
WO2004022777A1 (fr) * 2002-09-04 2004-03-18 Johnson & Johnson Research Pty Ltd Procedes d'utilisation d'adnds dans la mediation d'arn interferents (arni)
WO2004024872A2 (fr) * 2002-09-13 2004-03-25 California Institute Of Technology Methode d'expression de petites molecules d'arn antiviral a l'interieur d'une cellule

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KAYKAS AJAMETE ET AL: "A plasmid-based system for expressing small interfering RNA libraries in mammalian cells", BMC CELL BIOLOGY, vol. 5, no. April 30, 30 April 2004 (2004-04-30), XP002299176, ISSN: 1471-2121 *
SCHERR M ET AL: "GENE SILENCING MEDIATED BY SMALL INTERFERING RNAS IN MAMMALIAN CELLS", CURRENT MEDICINAL CHEMISTRY, BENTHAM SCIENCE PUBLISHERS BV, BE, vol. 10, no. 3, February 2003 (2003-02-01), pages 245 - 256, XP009019025, ISSN: 0929-8673 *
TRAN NHAM ET AL.: "Expressing functional siRNAs in mammalian cells using convergent transcription.", BMC BIOTECHNOLOGY, vol. 3, no. 21 Cited November 24, 2003, 6 November 2003 (2003-11-06), XP002299175, ISSN: 1472-6750 *
ZHANG J. ET AL.: "Targeted gene silencing by small interfering RNA-base knock-down technology", CURRENT PHARMACEUTICAL BIOTECHNOLOGY, vol. 5, no. 1, February 2004 (2004-02-01), pages 1 - 7, XP001183838 *
ZHENG LIANXING ET AL.: "An approach to genomewide screens of expressed small interfering RNAs in mammalian cells.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 101, no. 1, 6 January 2004 (2004-01-06), pages 135 - 140, XP002299174, ISSN: 0027-8424 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006033756A3 (fr) * 2004-08-23 2007-01-18 Nucleonics Inc Constructions d'expression de promoteurs d'arn polymerase iii multiples
US7985581B2 (en) 2004-08-23 2011-07-26 Alnylam Pharmaceuticals, Inc. Multiple RNA polymerase III promoter expression constructs
WO2009071722A1 (fr) * 2007-12-07 2009-06-11 Newbiotechnic, S.A. PROCÉDÉS ET TROUSSES POUR LA PRÉPARATION DE GÉNOTHÈQUES D'ARNsi SPÉCIFIQUES D'UN TRANSCRIPTOME PAR TRANSCRIPTION CONVERGENTE

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