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WO1998005771A1 - Procede pour accroitre l'effet d'arn antisens dans des cellules - Google Patents

Procede pour accroitre l'effet d'arn antisens dans des cellules Download PDF

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Publication number
WO1998005771A1
WO1998005771A1 PCT/DE1997/001692 DE9701692W WO9805771A1 WO 1998005771 A1 WO1998005771 A1 WO 1998005771A1 DE 9701692 W DE9701692 W DE 9701692W WO 9805771 A1 WO9805771 A1 WO 9805771A1
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WO
WIPO (PCT)
Prior art keywords
cells
rnase
sense rna
expression
vector
Prior art date
Application number
PCT/DE1997/001692
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German (de)
English (en)
Inventor
Dieter Werner
Christof Granzow
Marie Schubert
Karsten Rothbarth
Gunnar Dittmar
Herrmann Stammer
Ivan Todorov
Original Assignee
Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
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.)
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Publication date
Application filed by Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts filed Critical Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Publication of WO1998005771A1 publication Critical patent/WO1998005771A1/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
    • C12N15/113—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
    • 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 [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present invention relates to a method for increasing the activity of anti-sense RNA in cells and to a means for carrying out the method.
  • New techniques for inhibiting gene expression often involve the use of anti-sense RNA.
  • This is an RNA that is complementary to and binds to regions of the mRNA of a gene.
  • a duplex molecule is formed that is not translated by the mRNA. An inhibition of gene expression can thus be achieved.
  • duplex molecule is often not stable, i.e. the mRNA becomes free for translation again, whereby the inhibition of gene expression is weak or does not occur at all.
  • the present invention is therefore based on the object of providing a means with which a strong inhibition of gene expression can be achieved.
  • this is achieved by a method for increasing the activity of anti-sense RNA in cells, which comprises the expression of a (ds) RNAse in the cells containing the anti-sense RNA.
  • anti-sense RNA encompasses any RNA molecule which is suitable as anti-sense RNA, ie is complementary to regions of an RNA, in particular mRNA and very particularly regulatory elements thereof, and by binding to these regions inhibits the Gene expression.
  • the anti-sense RNA can also include DNA sequences.
  • the anti-sense RNA can be present as such or in the form of a vector or vector construct encoding it, which is sometimes also referred to as "minigen".
  • minigen a vector can be a common expression vector. It may be favorable if the expression of the sequence coding for the anti-sense RNA is under the control of a constitutive or inducible promoter, such as a tissue- or tumor-specific see promoter, stands.
  • enhancement of activity indicates that the action of an anti-sense RNA, i.e. inhibition of gene expression is increased. According to the invention this is achieved in that duplex molecules from mRNA and anti
  • Sense RNA can be degraded by a (ds) RNAse and thus only a reduced portion of the mRNA can be translated.
  • (ds) RNAse encompasses any RNAse that can recognize and degrade double-stranded RNA.
  • a (ds) RNAse is found e.g. in the yeast strain
  • the (ds) RNAse can be present as such or in the form of a vector encoding it.
  • a vector can be a common expression vector. It can be advantageous if the expression of the sequence coding for the (ds) RNAse is under the control of a constitutive or inducible promoter, such as a tissue- or tumor-specific one
  • cells includes any cells in which an anti-sense RNA acts, i.e. can inhibit gene expression.
  • Examples of such cells are plant and animal, especially mammalian and very particularly human cells.
  • the cells can be in or outside an organism. The latter can be freshly isolated or kept in culture.
  • RNAse if it is in the form of a vector encoding it. However, if it lies as such, i.e. as a protein, techniques such as
  • RNAse Lipofection.
  • the presence of the anti-sense RNA or (ds) RNAse in the cells can be demonstrated by conventional methods. These are, for example Southern and Northern blot for nucleic acids, while Western blot and functional analyzes (eg determination of enzyme activity) are to be regarded as suitable for proteins.
  • the present invention further relates to cells which have a (ds)
  • RNAse Such cells can be obtained by conventional methods. It is expedient to transfect cells as defined above with a vector coding for a (ds) RNAse. This can be done using conventional transfection techniques, such as electroporation. The vector can remain episomal or integrate into the genome of the cells. The expression of the (ds) RNAse can therefore be transposed or stable, the latter being preferred.
  • Cells which express a (ds) RNAse represent a means of carrying out the method for increasing the action of anti-sense RNA in cells. Furthermore, they represent a system for designing and testing the action of anti-sense RNAs.
  • Another object of the present invention is a combination of an anti-sense RNA and a (ds) RNAse.
  • the anti-sense RNA can exist as such or in the form of a vector encoding it.
  • the (ds) RNAse can be present as such or in the form of a vector encoding it. It may be advantageous if the combination consists in the presence of a vector which codes for both the anti-sense RNA and for the (ds) RNAse.
  • a combination of an anti-sense RNA and a (ds) RNAse is suitable as a means of carrying out the method for increasing the action of anti-sense RNA in cells.
  • the present invention it is possible to increase the effect of an anti-sense RNA in cells. A strong inhibition of the expression of the corresponding gene can thus be achieved.
  • the present invention is thus widely used in medicine.
  • the present invention makes it possible to find new inhibitors of gene expression and to test their effect.
  • 1 shows the increase in activity of anti-sense RNA in cells.
  • (1) is the expression rate of the CAT gene without an anti-sense RNA.
  • (2) is the rate of expression of the CAT gene with an anti-sense RNA.
  • (3) is the rate of expression of the CAT gene with an anti-sense RNA and a (ds) RNAse.
  • Example 1 Production of expression vectors which contain the chloramphenicol acetyl transferase (CAT) gene in the 5 ' ⁇ 3' or 3'- 5 'direction.
  • CAT chloramphenicol acetyl transferase
  • the CAT gene was isolated from a conventional CAT vector and into the "multiple cloning site" of the expression vector pJ3 ⁇ (cf. Nucleic acids res. 18, (1990),
  • the insertion was in the 5 ' ⁇ 3' direction and the expression vector pJ3 ⁇ -CAT was obtained. In the other case, the insertion was carried out in the 3 '- »5' direction and the expression vector pJ3 ⁇ -TAC was obtained.
  • Example 2 Preparation of an expression vector which codes for a (ds) RNAse.
  • the gene coding for a (ds) RNAse was converted from a common genomic library of Schizosaccharomyces pombe by means of PCR amplification
  • pad + isolated.
  • primers which were derived from the known sequence of the pad + gene (cf. database: embl: S78982) were.
  • the pad + gene was cloned in the known vector pBluescript and confirmed by sequencing. After cloning into the usual expression vector pcDNA3 (InVitrogen), the expression vector pcDNA3-pad + was obtained.
  • Ehrlich ascites tumor cells (10 7 cells / ml) were transfected with the expression vectors pJ3 ⁇ -CAT, pJ3 ⁇ -TAC and pcDNA3-pad + (see Table 1). The transfection was carried out by means of electroporation (366V / 950 ⁇ F / electrode spacing
  • FIG. 1 It can be seen from FIG. 1 that an inhibition of the expression of CAT is obtained by transfection of pJ3 ⁇ -TAC (cf. FIG. 1 (2)). This is significantly increased if pJ3 ⁇ -TAC and pcDNA3-pad + are cotransfected (see Fig. 1 (3). It is thus clear that an increase in the activity of anti-sense RNA in cells can be obtained by the expression of a (ds) RNAse.

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

Abstract

L'invention concerne un procédé pour accroître l'effet d'ARN antisens dans des cellules, ledit procédé consistant à exprimer une ribonucléase (bicaténaire) dans les cellules contenant l'ARN antisens. L'invention concerne en outre des cellules exprimant une ribonucléase (bicaténaire), et une combinaison constituée d'un ARN antisens et d'une ribonucléase (bicaténaire) qui sont codés par un ou plusieurs vecteurs.
PCT/DE1997/001692 1996-08-07 1997-08-05 Procede pour accroitre l'effet d'arn antisens dans des cellules WO1998005771A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE1996131918 DE19631918A1 (de) 1996-08-07 1996-08-07 Steigerung der Wirkung von Anti-Sinn-RNA in Zellen
DE19631918.8 1996-08-07

Publications (1)

Publication Number Publication Date
WO1998005771A1 true WO1998005771A1 (fr) 1998-02-12

Family

ID=7802055

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE1997/001692 WO1998005771A1 (fr) 1996-08-07 1997-08-05 Procede pour accroitre l'effet d'arn antisens dans des cellules

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DE (1) DE19631918A1 (fr)
WO (1) WO1998005771A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0592685A1 (fr) * 1992-04-17 1994-04-20 Kirin Beer Kabushiki Kaisha Vegetal resistant a au moins deux virus et sa preparation

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5583032A (en) * 1992-10-21 1996-12-10 The Cleveland Clinic Foundation And National Institutes Of Health Method of cleaving specific strands of RNA

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0592685A1 (fr) * 1992-04-17 1994-04-20 Kirin Beer Kabushiki Kaisha Vegetal resistant a au moins deux virus et sa preparation

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BLOMBERG P ET AL: "Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III.", EMBO JOURNAL, (1990 JUL) 9 (7) 2331-40., XP002049389 *
GERDES K ET AL: "Mechanism of killer gene activation. Antisense RNA-dependent RNase III cleavage ensures rapid turn-over of the stable hok, srnB and pndA effector messenger RNAs.", JOURNAL OF MOLECULAR BIOLOGY, (1992 AUG 5) 226 (3) 637-49., XP002049386 *
HELENE C ET AL: "LA STRATEGIE ANTISENS: NOUVELLES APPROCHES THERAPEUTIQUES", MEDECINE SCIENCES, vol. 10, no. 3, 1 March 1994 (1994-03-01), pages 253 - 273, XP000576223 *
JIANG, C.-Z. ET AL.: "Destabilisation of rbcS sense transcripts by antisense RNA", PLANT MOLECULAR BIOLOGY., vol. 25, 1994, DORDRECHT NL, pages 569 - 576, XP002049387 *
NELLEN, W. & LICHTENSTEIN, C.: "WHAT MAKES AN MESSENGER-RNA ANTI-SENSE-ITIVE ?", TRENDS IN BIOCHEMICAL SCIENCES, (NOV 1993) VOL. 18, NO. 11, PP. 419-423., XP002049385 *
ROTONDO, G. & FRENDEWEY, D.: "PURIFICATION AND CHARACTERIZATION OF THE PAC1 RIBONUCLEASE OF SCHIZOSACCHAROMYCES-POMBE", NUCLEIC ACIDS RESEARCH, (15 JUN 1996) VOL. 24, NO. 12, PP. 2377-2386., XP002049388 *

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Publication number Publication date
DE19631918A1 (de) 1998-02-12

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