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DE19631919C2 - Anti-sense RNA with secondary structure - Google Patents

Anti-sense RNA with secondary structure

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DE19631919C2
DE19631919C2 DE1996131919 DE19631919A DE19631919C2 DE 19631919 C2 DE19631919 C2 DE 19631919C2 DE 1996131919 DE1996131919 DE 1996131919 DE 19631919 A DE19631919 A DE 19631919A DE 19631919 C2 DE19631919 C2 DE 19631919C2
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sense rna
vector
expression
secondary structure
gene
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DE19631919A1 (en
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Dieter Prof Dipl Chem D Werner
Christof Dr Granzow
Gaby Dipl Biol Joswig
Karsten Dipl Chem Dr Rothbarth
Marie Schubert
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Deutsches Krebsforschungszentrum DKFZ
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Deutsches Krebsforschungszentrum DKFZ
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Priority to DE1996131919 priority Critical patent/DE19631919C2/en
Priority to PCT/DE1997/001691 priority patent/WO1998005770A2/en
Priority to EP97936610A priority patent/EP0918853A2/en
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    • C—CHEMISTRY; METALLURGY
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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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    • 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
    • C12N15/1137—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 against enzymes
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    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01028—Chloramphenicol O-acetyltransferase (2.3.1.28)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

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Description

Die vorliegende Erfindung betrifft eine Anti-Sinn-RNA mit Sekundärstruktur, eine sie enthaltende Kombination sowie die Verwendung beider.The present invention relates to an anti-sense RNA with a secondary structure, a combination containing them and the use of both.

WO 94/12633 beschreibt bestimmte Anti-Sinn-Sequenzen von Nukleotidbasen, die an einem oder beiden Enden der Anti-Sinn-Sequenz weitere Nukleotide besitzen, die eine Sekundärstruktur bilden.WO 94/12633 describes certain anti-sense sequences of nucleotide bases that have at one or both ends of the anti-sense sequence further nucleotides which form a secondary structure.

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 to inhibit gene expression often involve use of anti-sense RNA. This is an RNA that targets areas of a gene's mRNA is complementary and binds to them. A duplex molecule is formed, which the Translation of the mRNA is withdrawn. This can inhibit gene expression can 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 Gen­ expression schwach ist oder gar nicht eintritt.However, it has been shown that the duplex molecule is often not stable, i. H. The mRNA becomes free for translation again, thereby inhibiting the 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 eine Anti-Sinn-RNA mit besonderen Sekundär­ strukturen erreicht, wobei die Anti-Sinn-RNA in Form eines sie kodierenden Vektors vorliegt.According to the invention, this is achieved by an anti-sense RNA with a special secondary structures achieved, the anti-sense RNA in the form of a them coding vector is present.

Mit dem Ausdruck "besonderer Sekundärdruck" ist gemeint, daß es sich nicht um eine natürlich vorkommende Sekundärstruktur handelt, sondern daß diese künstlich erzeugt worden ist.By the term "special secondary pressure" it is meant that it is not is a naturally occurring secondary structure, but that it is artificial has been generated.

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. Erfindungsgemäß liegt die Anti-Sinn-RNA in Form eines sie kodierenden Vektors vor. 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 konstitutiven oder induzierbaren Promotors, wie eines Gewebe- oder Tumor-spezifischen Promotors, steht.The term "anti-sense RNA" includes any RNA molecule that acts as an anti Sinn RNA is suitable, i. H. complementary to areas of an RNA, in particular mRNA  and especially regulatory elements of this, is and by binding to it Areas causes an inhibition of gene expression. The anti-sense RNA can too Include DNA sequences. According to the invention, the anti-sense RNA is in shape of a vector encoding them. Such a vector can be a common one Expression vector. It can be favorable if the expression of the for the Anti-sense RNA coding sequence under the control of a constitutive or inducible promoter, such as a tissue- or tumor-specific promoter, stands.

Der Ausdruck "Sekundärstruktur" umfaßt jegliche DNA- und/oder RNA-Sequenz, die in einer Anti-Sinn-RNA vorliegen kann und eine zumindest teilweise "Hairpin"- Struktur aufweist, d. h. einzelne Basenpaare unterliegen einer Rückfaltung. Die Sekundärstruktur kann innerhalb der Anti-Sinn-RNA vorliegen. Auch kann sie am 5'- und/oder 3'-Ende der Anti-Sinn-RNA vorliegen. Liegen mehrere Sekundär­ strukturen vor, können diese gleich oder verschieden voneinander sein. Vorzugs­ weise ist die Sekundärstruktur eine (GC)n-Palindrom-(GC)n-, (AT)n-Palindrom-(AT)n-, oder (CG)n-Palindrom-(CG)n-Sequenz, wobei es besonders bevorzugt ist, wenn n=20 und das Palindrom eine EcoRI-Restriktionsstelle ist. Bevorzugt sind auch komplizierte Palindrome wie (AGCT)n oder (GAATTC)n.The term "secondary structure" encompasses any DNA and / or RNA sequence which can be present in an anti-sense RNA and which has an at least partially "hairpin" structure, ie individual base pairs are subject to refolding. The secondary structure can exist within the anti-sense RNA. It can also be present at the 5 'and / or 3' end of the anti-sense RNA. If there are several secondary structures, they can be the same or different from one another. Preference, the secondary structure is a (GC) n -Palindrom- (GC) n -, (AT) n -Palindrom- (AT) n -, or (CG) n -Palindrom- (CG) n sequence, wherein particularly is preferred if n = 20 and the palindrome is an EcoRI restriction site. Complicated palindromes such as (AGCT) n or (GAATTC) n are also preferred.

Eine erfindungsgemäße Anti-Sinn-RNA kann durch übliche Verfahren hergestellt werden. Günstig ist es, durch Oligonukleotidsynthese eine doppelsträngige (GC)20- EcoRI-(GC)20-Sequenz herzustellen und diese an das 5'-Ende der cDNA-Sequenz eines zu hemmenden Gens zu ligieren. Das erhaltene DNA-Molekül wird in 3'→5, Richtung an den Promotor eines Vektors ligiert. Der erhaltene Vektor führt zur Expression der erfindungsgemäßen Anti-Sinn-RNA. Ergänzend wird auf Sambrook, Fritsch, Maniatis, A Laboratory Mannual, Cold Spring Harbor Laboratory Press, 1989, verwiesen.An anti-sense RNA according to the invention can be produced by customary methods. It is favorable to prepare a double-stranded (GC) 20 - EcoRI (GC) 20 sequence by oligonucleotide synthesis and to ligate this to the 5 'end of the cDNA sequence of a gene to be inhibited. The DNA molecule obtained is ligated in 3 '→ 5 direction to the promoter of a vector. The vector obtained leads to the expression of the anti-sense RNA according to the invention. In addition, reference is made to Sambrook, Fritsch, Maniatis, A Laboratory Mannual, Cold Spring Harbor Laboratory Press, 1989.

Eine erfindungsgemäße Anti-Sinn-RNA kann in Form eines sie kodierenden Vektors in Zellen eingebracht werden. Die Zellen können jegliche Zellen, wie Pflanzen- und tierische, insbesondere Säugetier- und ganz besonders menschliche Zellen, sein. Die Zellen können innerhalb eines Organismus oder außerhalb eines solchen vorliegen. Letztere können frisch isoliert oder in Kultur gehalten sein. Das Einbringen der Anti-Sinn-RNA in die Zellen kann durch übliche Transfektionstechniken, wie Elektroporation, erfolgen.An anti-sense RNA according to the invention can be in the form of a coding vector are introduced into cells. The cells can do any Cells, such as plant and animal, especially mammalian, and very particularly  human cells. The cells can be inside an organism or outside of one. The latter can be freshly isolated or in culture be kept. The introduction of the anti-sense RNA into the cells can be done by conventional means Transfection techniques such as electroporation take place.

Ein weiterer Gegenstand der vorliegenden Erfindung ist eine Kombination aus einer erfindungsgemäßen Anti-Sinn-RNA und einer (ds)RNAse. Dies ist eine RNAse, die doppelsträngige RNA erkennen und abbauen kann. Eine (ds)RNAse findet sich z. B. in dem Hefestamm Schizosaccharomyces pombe (pac1+). In der Kombination kann die erfindungsgemäße Anti-Sinn-RNA als solche oder in Form eines sie kodierenden Vektors vorliegen. Ebenso 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 konstitutiven oder induzierbaren Promotors, wie eines Gewebe- oder Tumor-spezifischen Promotors, steht. Ferner kann es von Vorteil sein, wenn die Kombination darin besteht, daß ein Vektor vorliegt, der sowohl für die erfindungsgemäße Anti-Sinn-RNA als auch für die (ds)RNAse kodiert. Hinsichtlich des Vektors wird auf vorstehende Aus­ führungen verwiesen.Another object of the present invention is a combination of anti-sense RNA according to the invention and a (ds) RNAse. This is an RNAse that can recognize and break down double-stranded RNA. A (ds) RNAse is found e.g. B. in the yeast strain Schizosaccharomyces pombe (pac1 +). In combination the anti-sense RNA according to the invention as such or in the form of it coding vector are present. Likewise, the (ds) RNAse as such or in form of a vector encoding them. Such a vector can be a common one Expression vector. It can be favorable if the expression of the for the (ds) RNAse coding sequence under the control of a constitutive or inducible promoter, such as a tissue- or tumor-specific promoter, stands. It may also be advantageous if the combination is that a vector is present which is both for the anti-sense RNA according to the invention and encoded for the (ds) RNAse. Regarding the vector, reference is made to the above guided tours.

Die Kombination aus einer erfindungsgemäßen Anti-Sinn-RNA und einer (ds)RNAse kann in Zellen eingebracht werden. Hinsichtlich der Zellen und des Einbringens der Anti-Sinn-RNA wird auf vorstehende Ausführungen verwiesen. Die (ds)RNAse kann als solche, d. h. als Protein, durch übliche Verfahren, wie Lipofektion, eingebracht werden. In Form eines sie kodierenden Vektors kann die (ds)RNAse durch Ver­ fahren eingebracht werden, wie sie für die Anti-Sinn-RNA genannt wurden.The combination of an anti-sense RNA according to the invention and a (ds) RNAse can be introduced into cells. Regarding the cells and the introduction of the Anti-sense RNA is referred to the above explanations. The (ds) RNAse can as such, d. H. as a protein, introduced by conventional methods such as lipofection will. In the form of a vector encoding it, the (ds) RNAse can be determined by Ver drive, as they were called for the anti-sense RNA.

Die vorliegende Erfindung stellt eine Anti-Sinn-RNA und eine sie enthaltende Kombination bereit, die eine starke Hemmung der Genexpression bewirken. Die vorliegende Erfindung findet somit eine breite Anwendung in der Molekularbiologie und der Medizin. Insbesondere kann an die Diagnose und/oder Therapie von Erkrankungen gedacht werden, bei denen einzelne Proteine auslösend oder ver­ stärkend sind. Dies sind z. B. Erkrankungen, bei denen Hormone eine große Rolle spielen, Tumorerkrankungen und virale Infektionen, wie HIV und AIDS.The present invention provides an anti-sense RNA and one containing it Combination ready, which cause a strong inhibition of gene expression. The The present invention is thus widely used in molecular biology and medicine. In particular, the diagnosis and / or therapy of Diseases are thought in which individual proteins trigger or ver  are strengthening. These are e.g. B. Diseases in which hormones play a large role play tumor diseases and viral infections such as HIV and AIDS.

Kurze Beschreibung der ZeichnungBrief description of the drawing

Fig. 1 zeigt die Hemmung der Genexpression durch eine erfindungsgemäße Anti-Sinn-RNA. (1) ist die Expressionsrate des CAT-Gens in Anwe­ senheit einer Anti-Sinn-RNA. (2) ist die Expressionsrate des CAT- Gens in Anwesenheit einer Anti-Sinn-RNA mit Sekundärstruktur I. (3) ist die Expressionsrate des CAT-Gens in Anwesenheit einer Anti-Sinn- RNA mit Sekundärstruktur II. Fig. 1 shows the inhibition of gene expression by an inventive anti-sense RNA. (1) is the rate of expression of the CAT gene in the presence of an anti-sense RNA. (2) is the rate of expression of the CAT gene in the presence of an anti-sense RNA with secondary structure I. (3) is the rate of expression of the CAT gene in the presence of an anti-sense RNA with secondary structure II.

Fig. 2 zeigt die Hemmung der Genexpression durch eine erfindungsgemäße Anti-Sinn-RNA. (1) ist die Expressionsrate des CAT-Gens in Anwe­ senheit einer Anti-Sinn-RNA mit Sekundärstruktur I. (2) ist die Ex­ pressionsrate des CAT-Gens in Anwesenheit einer Anti-Sinn-RNA mit Sekundärstruktur I und einer (ds)RNAse. Fig. 2 shows the inhibition of gene expression by an inventive anti-sense RNA. (1) is the expression rate of the CAT gene in the presence of an anti-sense RNA with secondary structure I. (2) is the expression rate of the CAT gene in the presence of an anti-sense RNA with secondary structure I and a (ds) RNAse.

Die Erfindung wird durch die nachfolgenden Beispiele erläutert.The invention is illustrated by the following examples.

Beispiel 1example 1 Herstellung von Expressions-Vektoren, die das Chloramphenicolace­ tyltransferase (CAT)-Gen in 5'→3' bzw. 3'→5' Richtung enthaltenProduction of expression vectors representing the chloramphenicolace tyltransferase (CAT) gene in 5 '→ 3' or 3 '→ 5' direction included

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), 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. The CAT gene was isolated from a common CAT vector and in the "multiple cloning site" of the expression vector pJ3Ω (cf. Nucleic acids res. 18, (1990), 1068). In one case the Insertion in 5 '→ 3' direction and it became the expression vector Obtained pJ3Ω-CAT. In the other case, the insertion took place in 3 '→ 5' Direction and the expression vector pJ3Ω-TAC was obtained.  

Beispiel 2Example 2 Herstellung von Expressionsvektoren, die das CAT-Gen in 3'→5' Richtung und eine für eine Sekundärstruktur I bzw. II kodierende Sequenz enthaltenGeneration of expression vectors which the CAT gene in 3 '→ 5' Direction and a coding for a secondary structure I or II Sequence included (A) Expressionsvektor mit einer (GC)20-EcoRI-(GC)20-Sequenz am 5'-Ende des CAT-Gens (Sekundärstruktur I)(A) Expression vector with a (GC) 20 EcoRI (GC) 20 sequence at the 5 'end of the CAT gene (secondary structure I) 1. Herstellung einer (GC)20-EcoRI-(GC)20-Sequenz1. Preparation of a (GC) 20 EcoRI (GC) 20 sequence

  • (a) Mittels eines automatischen Synthese-Geräts (Oligonukleotid- Synthesizer) wurden 2 Oligodesoxynukleotide hergestellt:
    (a) 2 oligodeoxynucleotides were produced using an automatic synthesis device (oligonucleotide synthesizer):
  • (b) Die beiden Oligodesoxynukleotide wurden im Verhältnis 1 : 1 gemischt, auf 90°C erhitzt, danach langsam unter "annea­ ling"-Bedingungen auf Raumtemperatur abgekühlt. Dabei ent­ stand ein DNA Doppelstrang folgender Struktur:
    (b) The two oligodeoxynucleotides were mixed in a ratio of 1: 1, heated to 90 ° C., then slowly cooled to room temperature under "annealing" conditions. This resulted in a DNA double strand with the following structure:
  • (c) Unter Ligationsbedingungen entstanden Vielfache der in (b) beschriebenen DNA
    (c) Multiples of the DNA described in (b) were generated under ligation conditions
  • (d) Die Ligationsprodukte wurden durch Gelelektrophorese nach Größe aufgetrennt und eine Sequenz, bestehend aus Dimeren, wurde aus dem Gel eluiert und mittels Polynukleotidkinase /ATP phosphoryliert.
    (d) The ligation products were size-separated by gel electrophoresis and a sequence consisting of dimers was eluted from the gel and phosphorylated using polynucleotide kinase / ATP.
  • (e) Diese Sequenz wurde zunächst in die EcoRI-Stelle des üblichen Klonierungsvektors pBluescript (Stratagene) eingesetzt, aus dem sie durch geeignete Restriktionsenzyme zur Umklonierung in-den Vektor, der das CAT-Gen in 3'→ 5' Richtung aufweist, entnommen werden konnte.(e) This sequence was first inserted into the EcoRI site of the usual Cloning vector pBluescript (Stratagene) used, from by suitable restriction enzymes for recloning into the vector which has the CAT gene in the 3 '→ 5' direction, could be removed.
2. Einbau der (GC)20-EcoRI-(GC)20) Sequenz in den Vektor, der das CAT- Gen in 3'→5' Richtung aufweist2. Incorporation of the (GC) 20 -EcoRI- (GC) 20) sequence into the vector which has the CAT gene in the 3 '→ 5' direction

Der Vektor pJ3Ω-TAC von Beispiel 1 wurde in der "multiple cloning site" zwischen dem Promotor und der TAC-Insertion mit geeigneten Restriktionsenzymen geschnitten. Die (GC)20-EcoRI-(GC)20) Sequenz wurde mit den entsprechenden Enzymen aus dem pBluescript-Vektor von Beispiel 2(e) entnommen. Die beiden Nukleinsäuren wurden per Ligation verbunden. Es wurde der Expressionsvektor pJ3Ω-TAC-Sek. I erhalten.The vector pJ3Ω-TAC from Example 1 was cut in the "multiple cloning site" between the promoter and the TAC insert with suitable restriction enzymes. The (GC) 20 EcoRI (GC) 20) sequence with the appropriate enzymes was taken from the pBluescript vector from Example 2 (e). The two nucleic acids were linked by ligation. The expression vector pJ3Ω-TAC-sec. I received.

(B) Expressionsvektor mit einer (GC)20-EcoRI-(GC)20-Sequenz am 3'-Ende des CAT-Gens (Sekundärstruktur II)(B) Expression vector with a (GC) 20 EcoRI (GC) 20 sequence at the 3 'end of the CAT gene (secondary structure II)

Die unter Beispiel 2 (A) hergestellte (GC)20-EcoRI-(GC)20-Sequenz wurde in den Vektor pJ3Ω-TAC am 3'-Ende des TAC-Gens eingesetzt. Es wurde der Expressionsvektor pJ3Ω-TAC-Sek.II erhalten. The (GC) 20 EcoRI (GC) 20 sequence prepared in Example 2 (A) was inserted into the vector pJ3Ω-TAC at the 3 'end of the TAC gene. The expression vector pJ3Ω-TAC-sec. II was obtained.

Beispiel 3Example 3 Herstellung eines Expressionsvektors, der für eine (ds) RNAse kodiertGeneration 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 Gen (pac1+) isoliert. Hierzu wurden Primer verwendet, die aus der bekannten Sequenz des Gens pac1+ (vgl. Datenbank: embl: S78982) abgeleitet worden waren. Das Gen pac1+ wurde in dem bekannten Vektor pBluescript kloniert und durch Sequenzierung bestätigt. Nach Umklonierung in den üblichen Expressionsvektor pcDNA3 (InVitrogen) wurde der Expressionsvektor pcDNA3-pac1+ erhalten.From a common Schizosaccharomyces genomic library PCR was used to amplify pombe for (ds) RNAse coding gene (pac1 +) isolated. For this, primers were used from the known sequence of the pac1 + gene (see database: embl: S78982) had been derived. The pac1 + gene was found in cloned the known vector pBluescript and by sequencing approved. After cloning into the usual expression vector pcDNA3 (InVitrogen) became the expression vector pcDNA3-pac1 + receive.

Beispiel 4Example 4 Hemmung der Genexpression durch eine Anti-Sinn-RNA mit Sekun­ därstrukturInhibition of gene expression by an anti-sense RNA with seconds intestinal structure

  • (a) Ehrlich Ascites Tumorzellen (107 Zellen/ml) wurden mit den Expressionsvektoren pJ3Ω-CAT, pJ3Ω-TAC, pJ3Ω-TAC-Sek. I bzw. pJ3Ω-TAC-Sek. II transfiziert (vgl. Tabelle 1). Die Transfektion wurde mittels Elektroporation (366V/950yF/Ele­ ktrodenabstand D=4mm) durchgeführt. 24 h nach Transfek­ tion wurden die Zellen geerntet, lysiert und Aliquote mit radio­ aktiv markiertem Chloramphenicol inkubiert. Es wurde die Konversionsrate (in Ac-, Di-Ac-Chloramphenicol) nach DC durch Messung der Radioaktivität bestimmt.

    Tabelle 1

    (a) Ehrlich ascites tumor cells (10 7 cells / ml) were expressed with the expression vectors pJ3Ω-CAT, pJ3Ω-TAC, pJ3Ω-TAC-sec. I or pJ3Ω-TAC-sec. II transfected (see Table 1). The transfection was carried out by means of electroporation (366V / 950yF / electrode distance 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.

    Table 1

Aus Fig. 1 geht hervor, daß durch Transfektion von pJ3Ω-TAC-Sek. I bzw. pJ3Ω-TAC-Sek. II (vgl. Fig. 1, (2), (3) eine stärkere Hemmung der Expres­ sion des CAT-Gens erreicht werden kann, als wenn pJ3Ω-TAC (vgl. Fig. 1, (1) verwendet wird.From Fig. 1 it appears that by transfection of pJ3Ω-TAC-sec. I or pJ3Ω-TAC-sec. II (cf. Fig. 1, (2), (3) a greater inhibition of the expression of the CAT gene can be achieved than if pJ3Ω-TAC (cf. Fig. 1, (1) is used.

  • (b) Ehrlich Ascites Tumorzellen (10-7 Zellen/ml) wurden mit den Expres­ sionsvektoren pJ3Ω-CAT, pJ3Ω-TAC-Sek. I bzw. pcDNA3-pac1+ tranfiziert (vgl. Tabelle 2). Die Transfektionsbedingungen waren wie in Beispiel 4 (a) beschrieben.

    Tabelle 2

    (b) Ehrlich ascites tumor cells (10- 7 cells / ml) were expressed with the expression vectors pJ3Ω-CAT, pJ3Ω-TAC-Sek. I or pcDNA3-pac1 + transfected (see Table 2). The transfection conditions were as described in Example 4 (a).

    Table 2

Aus Fig. 2 geht hervor, daß durch Kotransfektion von pJ3Ω-TAC-Sek. I mit pcDNA3-pac1+ (vgl. Fig. 2 (2)) eine stärkere Hemmung der Expression von CAT erhalten wird, als wenn pJ3Ω-TAC-Sek. I (vgl. Fig. 2, (1) alleine ver­ wendet wird. From Fig. 2 it appears that by co-transfection of pJ3Ω-TAC-sec. I with pcDNA3-pac1 + (see FIG. 2 (2)) a stronger inhibition of the expression of CAT is obtained than if pJ3Ω-TAC-sec. I (see FIG. 2, (1) is used alone ver.

Somit wird deutliche daß eine Anti-Sinn-RNA mit Sekundärstruktur eine größere Hemmwirkung auf die Genexpression hat als eine Anti-Sinn-RNA ohne Sekundärstruktur. Ferner wird deutlich, daß die Hemmwirkung der Anti-Sinn-RNA mit Sekundärstruktur noch gesteigert werden kann, wenn zusätzlich zu gegebenenfalls natürlich vorhandenen (ds)RNAsen eine (ds)RNAse-Aktivität mittels der beschriebenen Verfahren hervorgerufen bzw. erzeugt wird.Thus it becomes clear that an anti-sense RNA with a secondary structure is a has a greater inhibitory effect on gene expression than an anti-sense RNA without secondary structure. It also becomes clear that the inhibitory effect of Anti-sense RNA with secondary structure can still be increased, though in addition to any naturally present (ds) RNAsen (ds) RNAse activity caused by the described methods or is produced.

Claims (7)

1. Anti-Sinn-RNA mit besonderen Sekundärstrukturen, wobei die Anti-Sinn- RNA in Form eines sie kodierenden Vektors vorliegt.1. Anti-sense RNA with special secondary structures, the anti-sense RNA is in the form of a vector encoding it. 2. Anti-Sinn-RNA nach Anspruch 1, dadurch gekennzeichnet, daß die Sekun­ därstruktur am 5'- und/oder 3'-Ende der Anti-Sinn-RNA geschaffen worden ist.2. Anti-sense RNA according to claim 1, characterized in that the seconds The structure at the 5 'and / or 3' end of the anti-sense RNA was created is. 3. Anti-Sinn-RNA nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß die Sekundärstruktur eine (GC)n-Palindrom-(GC)n- oder (CG)n-Palindrom-(CG)n- Sequenz ist.3. Anti-sense RNA according to claim 1 or 2, characterized in that the secondary structure is a (GC) n -palindrome- (GC) n - or (CG) n -palindrome- (CG) n - sequence. 4. Anti-Sinn-RNA nach Anspruch 3, dadurch gekennzeichnet, daß n = 20 und das Palindrom eine EcoRI-Restriktionsstelle ist.4. Anti-sense RNA according to claim 3, characterized in that n = 20 and the palindrome is an EcoRI restriction site. 5. Kombination, umfassend die Anti-Sinn-RNA nach einem der Ansprüche 1-4 und eine (ds)RNAse.5. Combination comprising the anti-sense RNA according to any one of claims 1-4 and a (ds) RNAse. 6. Kombination nach Anspruch 5, dadurch gekennzeichnet, daß die (ds)RNAse in Form eines sie kodierenden Vektors vorliegt.6. Combination according to claim 5, characterized in that the (ds) RNAse is in the form of a vector encoding it. 7. Verwendung der Anti-Sinn-RNA nach einem der Ansprüche 1-4 und der Kombination nach Anspruch 5 oder 6 zur Hemmung der Genexpression.7. Use of the anti-sense RNA according to any one of claims 1-4 and Combination according to claim 5 or 6 for inhibiting gene expression.
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