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WO1998005771A1 - Method for increasing the effect of antisense rna in cells - Google Patents

Method for increasing the effect of antisense rna in cells 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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cells
rnase
sense rna
expression
vector
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PCT/DE1997/001692
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German (de)
French (fr)
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Dieter Werner
Christof Granzow
Marie Schubert
Karsten Rothbarth
Gunnar Dittmar
Herrmann Stammer
Ivan Todorov
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Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

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  • 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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Abstract

The invention concerns a method for increasing the effect of antisense RNA in cells, said method comprising the expression of a (ds)RNAase in the cells containing antisense RNA. The invention also concerns cells which express a (ds)RNAase and a combination of an antisense RNA and a (ds)RNAase which are coded by one or several vectors.

Description

Steigerung der Wirkung von Anti-Sinn-RNA in Zellen Enhance the action of anti-sense RNA in cells
Die vorliegende Erfindung betrifft ein Verfahren zur Wirkungssteigerung von Anti-Sinn-RNA in Zellen sowie ein Mittel zur Durchführung des Verfahrens.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.
Neue Techniken zur Hemmung der Genexpression umfassen häufig den Einsatz von Anti-Sinn-RNA. Dies ist eine RNA, die zu Bereichen der mRNA eines Gens komplementär ist und an diese bindet. Es entsteht ein Duplexmolekül, das der Translation der mRNA entzogen ist. Damit kann eine Hemmung der Genexpression erreicht werden.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.
Es hat sich allerdings gezeigt, daß das Duplexmolekül häufig nicht stabil ist, d.h. die mRNA wird wieder frei für die Translation, wodurch die Hemmung der Genexpression schwach ist oder gar nicht eintritt.However, it has been shown that the 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.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzu- stellen, mit dem eine starke Hemmung der Genexpression erzielt werden kann.The present invention is therefore based on the object of providing a means with which a strong inhibition of gene expression can be achieved.
Erfindungsgemäß wird dies durch ein Verfahren zur Wirkungssteigerung von Anti-Sinn-RNA in Zellen erreicht, das die Expression einer (ds)RNAse in den die Anti-Sinn-RNA enthaltenden Zellen umfaßt.According to the invention, 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.
Der Ausdruck "Anti-Sinn-RNA" umfaßt jegliches RNA-Molekül, das sich als Anti- Sinn-RNA eignet, d.h. komplementär zu Bereichen einer RNA, insbesondere mRNA und ganz besonders Regulationselementen dieser, ist und durch Bindung an diese Bereiche eine Hemmung der Genexpression bewirkt. Die Anti-Sinn-RNA kann auch DNA-Sequenzen umfassen. Ferner kann die Anti-Sinn-RNA als solche oder in Form eines sie kodierenden Vektors bzw. Vektorkonstrukts, das gelegentlich auch als "Minigen" bezeichnet wird, vorliegen. Ein solcher Vektor kann ein üblicher Expressionsvektor sein. Günstig kann es sein, wenn die Expression der für die Anti-Sinn-RNA kodierenden Sequenz unter der Kontrolle eines kon- stitutiven oder induzierbaren Promotors, wie eines Gewebe- oder Tumor-spezifi- sehen Promotors, steht.The term “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. Furthermore, 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". Such 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.
Der Ausdruck "Wirkungssteigerung" weist darauf hin, daß die Wirkung einer Anti-Sinn-RNA, d.h. die Hemmung der Genexpression, gesteigert ist. Erfindungs- gemäß wird dies dadurch erreicht, daß Duplexmoleküle aus mRNA und Anti-The term "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
Sinn-RNA durch eine (ds)RNAse abgebaut werden und somit nur ein reduzierter Anteil der mRNA translatiert werden kann..Sense RNA can be degraded by a (ds) RNAse and thus only a reduced portion of the mRNA can be translated.
Der Ausdruck "(ds)RNAse" umfaßt jegliche RNAse, die doppelsträngige RNA erkennen und abbauen kann. Eine (ds)RNAse findet sich z.B. in dem HefestammThe term "(ds) RNAse" encompasses any RNAse that can recognize and degrade double-stranded RNA. A (ds) RNAse is found e.g. in the yeast strain
Schizosaccharomyces pombe (pad + Gen). Ferner kann die (ds)RNAse als solche oder in Form eines sie kodierenden Vektors vorliegen. Ein solcher Vektor kann ein üblicher Expressionsvektor sein. Günstig kann es sein, wenn die Expression der für die (ds)RNAse kodierenden Sequenz unter der Kontrolle eines konstituti- ven oder induzierbaren Promotors, wie eines Gewebe- oder Tumor-spezifischenSchizosaccharomyces pombe (pad + gene). Furthermore, the (ds) RNAse can be present as such or in the form of a vector encoding it. Such 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
Promotors, steht.Promoter, stands.
Der Ausdruck "Zellen" umfaßt jegliche Zellen, in denen eine Anti-Sinn-RNA wirken, d.h. die Genexpression hemmen kann. Beispiele solcher Zellen sind Pflanzen- und tierische, insbesondere Säugetier- und ganz besonders menschliche Zellen. Die Zellen können in einem Organismus oder außerhalb eines solchen vorliegen. Letztere können frisch isoliert oder in Kultur gehalten sein.The term "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.
Der Ausdruck "Expression einer (ds)RNAse in Zellen, die eine Anti-Sinn-RNA enthalten" weist darauf hin, daß eine Anti-Sinn-RNA und eine (ds)RNAse inThe expression "expression of a (ds) RNAse in cells containing an anti-sense RNA" indicates that an anti-sense RNA and a (ds) RNAse in
Zellen eingeführt werden. Dies kann durch übliche Verfahren erfolgen. Liegt die Anti-Sinn-RNA als solche oder in Form eines sie kodierenden Vektors vor, eignen sich z.B. Transfektionstechniken, wie Elektroporation. Gleiches gilt für die (ds)RNAse, wenn sie in Form eines sie kodierenden Vektors vorliegt. Liegt sie allerdings als solche, d.h. als Protein vor, sind insbesondere Techniken, wieCells are introduced. This can be done by conventional methods. If the anti-sense RNA is present as such or in the form of a vector encoding it, e.g. Transfection techniques, such as electroporation. The same applies to the (ds) 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
Lipofektion, zu nennen. Das Vorliegen der Anti-Sinn-RNA bzw. der (ds)RNAse in den Zellen kann durch übliche Verfahren nachgewiesen werden. Dies sind z.B. Southern- und Northern-Blot für Nukleinsäuren, während Western-Blot und funktionelle Analysen (z.B. Enzymaktivitätsbestimmung) für Proteine als geeignet anzusehen sind.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.
Ein weiterer Gegenstand der vorliegenden Erfindung sind Zellen, die eine (ds)The present invention further relates to cells which have a (ds)
RNAse exprimieren. Solche Zellen können durch übliche Verfahren erhalten werden. Günstig ist es, Zellen, wie vorstehend definiert, mit einem für eine (ds)RNAse kodierenden Vektor zu transfizieren. Dies kann durch übliche Trans- fektionstechniken, wie Elektroporation, erfolgen. Der Vektor kann episomal verbleiben oder in das Genom der Zellen integrieren. Die Expression der (ds)RNA- se kann daher transiert oder stabil sein, wobei letzteres bevorzugt ist. Zellen, die eine (ds)RNAse exprimieren, stellen ein Mittel zur Durchführung des Verfahrens zur Wirkungssteigerung von Anti-Sinn-RNA in Zellen dar. Ferner stellen sie ein System dar, Anti-Sinn-RNAs zu konzipieren und in ihrer Wirkung zu testen.Express 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.
Ein weiterer Gegenstand der vorliegenden Erfindung ist eine Kombination aus einer Anti-Sinn-RNA und einer (ds)RNAse. Die Anti-Sinn-RNA kann als solche oder in Form eines sie kodierenden Vektors vorliegen. Ebenso kann die (ds)RNA- se als solche oder in Form eines sie kodierenden Vektors vorliegen. Günstig kann es sein, wenn die Kombination darin besteht, daß ein Vektor vorliegt, der sowohl für die Anti-Sinn-RNA als auch für die (ds)RNAse kodiert. Hinsichtlich des Vektors wird auf vorstehende Ausführungen verwiesen. Eine Kombination aus einer Anti-Sinn-RNA und einer (ds)RNAse eignet sich als Mittel zur Durchführung des Verfahrens zur Wirkungssteigerung von Anti-Sinn-RNA in Zellen.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. Likewise, 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. With regard to the vector, reference is made to the above statements. 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.
Mit der vorliegenden Erfindung ist es möglich, die Wirkung einer Anti-Sinn-RNA in Zellen zu steigern. Damit kann eine starke Hemmung der Expression des entsprechenden Gens erreicht werden. Die vorliegende Erfindung findet somit eine breite Anwendung in der Medizin. Insbesondere kann an die Diagnose und/oder Therapie von Erkrankungen gedacht werden, bei denen einzelne Proteine auslösend oder verstärkend sind. Dies sind z.B. Erkrankungen, bei denen Hormone eine große Rolle spielen, Tumorerkrankungen und virale Infektionen, wie HIV und AIDS. Desweiteren ermöglicht die vorliegende Erfindung, neue Inhibitoren der Genexpression zu finden und ihre Wirkung zu testen.With 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. In particular, one can think of the diagnosis and / or therapy of diseases in which individual proteins trigger or reinforce. These are, for example, diseases in which hormones play a major role, tumor diseases and viral infections, like HIV and AIDS. Furthermore, the present invention makes it possible to find new inhibitors of gene expression and to test their effect.
Kurze Beschreibung der ZeichnungBrief description of the drawing
Fig. 1 zeigt die Wirkungssteigerung von Anti-Sinn-RNA in Zellen. ( 1 ) ist die Expressionsrate des CAT-Gens ohne eine Anti-Sinn-RNA. (2) ist die Expressionsrate des CAT-Gens mit einer Anti-Sinn-RNA. (3) ist die Expressionsrate des CAT-Gens mit einer Anti-Sinn-RNA und einer (ds)RNAse.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.
Die Erfindung wird durch die nachfolgenden Beispiele erläutert.The invention is illustrated by the following examples.
Beispiel 1 : Herstellung von Expressions-Vektoren, die das Chloramphenicol- acetyltransferase (CAT)-Gen in 5'→ 3' bzw. 3'- 5' Richtung enthalten.Example 1: Production of expression vectors which contain the chloramphenicol acetyl transferase (CAT) gene in the 5 '→ 3' or 3'- 5 'direction.
Das CAT-Gen wurde aus einem üblichen CAT-Vektor isoliert und in die "multiple cloning site" des Expressionsvektors pJ3Ω (vgl. Nucleic acids res. 18, (1990),The CAT gene was isolated from a conventional CAT vector and into the "multiple cloning site" of the expression vector pJ3Ω (cf. Nucleic acids res. 18, (1990),
1068) inseriert. In einem Fall erfolgte die Insertion in 5'→3' Richtung und es wurde der Expressionsvektor pJ3Ω-CAT erhalten. Im anderen Fall erfolgte die Insertion in 3'-»5' Richtung und es wurde der Expressionsvektor pJ3Ω-TAC erhalten.1068). In one case the insertion was in the 5 '→ 3' direction and the expression vector pJ3Ω-CAT was obtained. In the other case, the insertion was carried out in the 3 '- »5' direction and the expression vector pJ3Ω-TAC was obtained.
Beispiel 2: Herstellung eines Expressionsvektors, der für eine (ds) RNAse kodiert.Example 2: Preparation of an expression vector which codes for a (ds) RNAse.
Aus einer üblichen genomischen Bibliothek von Schizosaccharomyces pombe wurde mittels einer PCR-Amplifikation das für eine (ds)RNAse kodierende GenThe gene coding for a (ds) RNAse was converted from a common genomic library of Schizosaccharomyces pombe by means of PCR amplification
(pad + ) isoliert. Hierzu wurden Primer verwendet, die aus der bekannten Sequenz des Gens pad + (vgl. Datenbank: embl: S78982) abgeleitet worden waren. Das Gen pad + wurde in dem bekannten Vektor pBluescript kloniert und durch Sequenzierung bestätigt. Nach Umklonierung in den üblichen Expressionsvektor pcDNA3 (InVitrogen) wurde der Expressionsvektor pcDNA3-pad + erhalten.(pad +) isolated. For this purpose, primers were used 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.
Beispiel 3: Steigerung der Wirkung von Anti-Sinn-RNA in ZellenExample 3: Increasing the Effect of Anti-Sense RNA in Cells
Ehrlich Ascites Tumorzellen ( 107 Zellen/ml) wurden mit den Expressionsvektoren pJ3Ω-CAT, pJ3Ω-TAC und pcDNA3-pad + transfiziert (vgl. Tabelle 1 ) . Die Transfektion wurde mittels Elektroporation (366V/950μF/ElektrodenabstandEhrlich ascites tumor cells (10 7 cells / ml) were transfected with the expression vectors pJ3Ω-CAT, pJ3Ω-TAC and pcDNA3-pad + (see Table 1). The transfection was carried out by means of electroporation (366V / 950μF / electrode spacing
D = 4mm) durchgeführt. 24 h nach Transfektion wurden die Zellen geerntet, lysiert und Aliquote mit radioaktiv markiertem Chloramphenicol inkubiert. Es wurde die Konversionsrate (in Ac-, Di-Ac-Chloramphenicol) nach DC durch Messung der Radioaktivität bestimmt.D = 4mm). 24 hours after transfection, the cells were harvested, lysed and aliquots were incubated with radioactively labeled chloramphenicol. The conversion rate (in Ac-, Di-Ac-Chloramphenicol) after DC was determined by measuring the radioactivity.
Tabelle 1 :Table 1 :
pJ3Ω-CAT 3 g 3μg 3 vgpJ3Ω-CAT 3 g 3μg 3 vg
pJ3Ω-TAC 7,5 yg 7,5/vgpJ3Ω-TAC 7.5 yg 7.5 / vg
pcDNA3-pad + - - 7,5μgpcDNA3-pad + - - 7.5μg
pJ3Ω 7,5 gpJ3Ω 7.5 g
pcDNA3 7,5y- g 7, 5μgpcDNA3 7.5y- 7.5 g
Figure imgf000007_0001
Figure imgf000007_0001
Aus Fig. 1 geht hervor, daß durch Transfektion von pJ3Ω-TAC eine Hemmung der Expression von CAT erhalten wird (vgl. Fig. 1 (2)) . Diese wird erheblich gesteigert, wenn pJ3Ω-TAC und pcDNA3-pad + kotransfiziert werden (vgl. Fig. 1 (3) . Somit wird deutlich, daß eine Wirkungssteigerung von Anti-Sinn-RNA in Zellen durch die Expression einer (ds) RNAse erhalten werden kann. It can be seen from FIG. 1 that an inhibition of the expression of CAT is obtained by transfection of pJ3Ω-TAC (cf. FIG. 1 (2)). This is significantly increased if pJ3Ω-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.

Claims

Patentansprüche claims
1 . Verfahren zur Wirkungssteigerung von Anti-Sinn-RNA in Zellen, umfassend die Expression einer (ds)RNAse in den die Anti-Sinn-RNA enthaltenden Zellen.1 . A method for increasing the activity of anti-sense RNA in cells, comprising the expression of a (ds) RNAse in the cells containing the anti-sense RNA.
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß die Zellen tierische Zellen sind.2. The method according to claim 1, characterized in that the cells are animal cells.
3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß die Anti-Sinn-RNA durch einen Vektor kodiert ist.3. The method according to claim 1 or 2, characterized in that the anti-sense RNA is encoded by a vector.
4. Verfahren nach einem der Ansprüche 1 - 3, dadurch gekennzeichnet, daß die (ds)RNAse durch einen Vektor kodiert ist.4. The method according to any one of claims 1-3, characterized in that the (ds) RNAse is encoded by a vector.
5. Verfahren nach einem der Ansprüche 3 oder 4, dadurch gekennzeichnet, daß die Anti-Sinn-RNA und die (ds)RNAse durch den gleichen Vektor kodiert sind.5. The method according to any one of claims 3 or 4, characterized in that the anti-sense RNA and the (ds) RNAse are encoded by the same vector.
6. Verfahren nach einem der Ansprüche 1 - 3, dadurch gekennzeichnet, daß die (ds)RNAse durch das Genom der Zellen kodiert ist.6. The method according to any one of claims 1-3, characterized in that the (ds) RNAse is encoded by the genome of the cells.
7. Zellen, die eine (ds)RNAse exprimieren.7. Cells expressing (ds) RNAse.
8. Zellen nach Anspruch 7, dadurch gekennzeichnet, daß die Zellen tierische Zellen sind.8. Cells according to claim 7, characterized in that the cells are animal cells.
9. Kombination aus einer Anti-Sinn-RNA und einer (ds)RNAse, wobei beide durch einen oder mehrere Vektoren kodiert sind. 9. Combination of an anti-sense RNA and a (ds) RNAse, both of which are encoded by one or more vectors.
PCT/DE1997/001692 1996-08-07 1997-08-05 Method for increasing the effect of antisense rna in cells WO1998005771A1 (en)

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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 *
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