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

Anti-sense RNA with secondary structure

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Publication number
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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anti
sense rna
secondary structure
vector
expression
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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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    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-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
    • C12N15/1137Non-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
    • CCHEMISTRY; METALLURGY
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    • C12YENZYMES
    • C12Y203/00Acyltransferases (2.3)
    • C12Y203/01Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01028Chloramphenicol O-acetyltransferase (2.3.1.28)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/11Antisense
    • C12N2310/111Antisense spanning the whole gene, or a large part of it
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/50Physical structure
    • C12N2310/53Physical structure partially self-complementary or closed

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 secondary structure, a combination containing them and to 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 antisense sequences of nucleotide bases having more nucleotides at one or both ends of the antisense sequence, forming a secondary structure.

Neue Techniken zur Hemmung der Genexpression umfassen häufig den Einsatz von Anti-Sinn-RNA. New techniques for inhibiting gene expression often include the use of anti-sense RNA. Dies ist eine RNA, die zu Bereichen der mRNA eines Gens komplementär ist und an diese bindet. This is an RNA that is complementary to portions of mRNA of a gene and binds to them. Es entsteht ein Duplexmolekül, das der Translation der mRNA entzogen ist. The result is a duplex molecule that is extracted from the translation of the mRNA. Damit kann eine Hemmung der Genexpression erreicht werden. Thus, inhibition of gene expression can be achieved.

Es hat sich allerdings gezeigt, daß das Duplexmolekül häufig nicht stabil ist, dh die mRNA wird wieder frei für die Translation, wodurch die Hemmung der Gen expression schwach ist oder gar nicht eintritt. However, it has shown that the duplex is unstable frequently, ie the mRNA is again free for translation, thereby inhibiting gene expression is weak or does not occur.

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 thus based on the object bereitzu provide a means by 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. this is achieved by a structure antisense RNA with special secondary According to the invention, the antisense RNA is present in the form of a vector encoding it.

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" is meant that it is not a naturally occurring secondary structure, but that it has been created artificially.

Der Ausdruck "Anti-Sinn-RNA" umfaßt jegliches RNA-Molekül, das sich als Anti- Sinn-RNA eignet, dh komplementär zu Bereichen einer RNA, insbesondere mRNA und ganz besonders Regulationselementen dieser, ist und durch Bindung an diese Bereiche eine Hemmung der Genexpression bewirkt. The term "antisense RNA" includes any RNA molecule, which is useful as anti-sense RNA, that is to regions of a RNA, especially mRNA, and most regulatory elements that is complementary, and by binding to these areas inhibition of gene expression causes. Die Anti-Sinn-RNA kann auch DNA-Sequenzen umfassen. The anti-sense RNA may also comprise DNA sequences. Erfindungsgemäß liegt die Anti-Sinn-RNA in Form eines sie kodierenden Vektors vor. According to the invention the anti-sense RNA is in the form of a vector encoding it. Ein solcher Vektor kann ein üblicher Expressionsvektor sein. Such a vector may be a conventional expression vector. 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. It may be favorable when the expression of the coding for the anti-sense RNA sequence under the control of a constitutive or inducible promoter, such as a tissue or tumor-specific promoter.

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, dh einzelne Basenpaare unterliegen einer Rückfaltung. The term "secondary structure" comprises any DNA and / or RNA sequence that can be present in an anti-sense RNA and an at least partially "hairpin" - has structure, ie single base pairs are subject to aliasing. Die Sekundärstruktur kann innerhalb der Anti-Sinn-RNA vorliegen. The secondary structure can be present within the anti-sense RNA. Auch kann sie am 5'- und/oder 3'-Ende der Anti-Sinn-RNA vorliegen. Also, they may be present at the 5'- and / or 3'-end of the anti-sense RNA. Liegen mehrere Sekundär strukturen vor, können diese gleich oder verschieden voneinander sein. If there are several secondary structures before, these can be identical or different. 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. Preferential manner, the secondary structure of a (GC) n -Palindrom- (GC) n -, (AT) n -Palindrom- (AT) n -, or (CG) n -Palindrom- (CG) n sequence, wherein particularly is preferred when n = 20 and the palindrome is an EcoRI restriction site. Bevorzugt sind auch komplizierte Palindrome wie (AGCT) n oder (GAATTC) n . Complicated as palindromes (AGCT) n or (GAATTC) n are preferred.

Eine erfindungsgemäße Anti-Sinn-RNA kann durch übliche Verfahren hergestellt werden. An inventive anti-sense RNA may be prepared by conventional methods. 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. Produce EcoRI (GC) 20 sequence and ligating it to the 5'-end of the cDNA sequence of a gene to be inhibited - It is advantageous by a double-stranded oligonucleotide (GC) 20 is. Das erhaltene DNA-Molekül wird in 3'→5, Richtung an den Promotor eines Vektors ligiert. The obtained DNA molecule is ligated into the 3 '→ 5, the direction to the promoter of a vector. Der erhaltene Vektor führt zur Expression der erfindungsgemäßen Anti-Sinn-RNA. The resulting vector leads to expression of anti-sense RNA to the invention. Ergänzend wird auf Sambrook, Fritsch, Maniatis, A Laboratory Mannual, Cold Spring Harbor Laboratory Press, 1989, verwiesen. We also refer 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. An inventive anti-sense RNA may be introduced in the form of a vector encoding them into cells. Die Zellen können jegliche Zellen, wie Pflanzen- und tierische, insbesondere Säugetier- und ganz besonders menschliche Zellen, sein. The cells can such as plant and animal, in particular mammalian and especially human cells be any cells. Die Zellen können innerhalb eines Organismus oder außerhalb eines solchen vorliegen. The cells can be present within an organism or outside of such. Letztere können frisch isoliert oder in Kultur gehalten sein. The latter can be fresh isolated or kept in culture. Das Einbringen der Anti-Sinn-RNA in die Zellen kann durch übliche Transfektionstechniken, wie Elektroporation, erfolgen. The introduction of the antisense RNA into the cells can be carried out by conventional transfection techniques such as electroporation.

Ein weiterer Gegenstand der vorliegenden Erfindung ist eine Kombination aus einer erfindungsgemäßen Anti-Sinn-RNA und einer (ds)RNAse. Another object of the present invention is a combination of an inventive anti-sense RNA and a (ds) RNAse. Dies ist eine RNAse, die doppelsträngige RNA erkennen und abbauen kann. This is an RNase that can detect double-stranded RNA and remove. Eine (ds)RNAse findet sich z. A (ds) RNase found, for. B. in dem Hefestamm Schizosaccharomyces pombe (pac1+). B. pombe in the yeast strain Schizosaccharomyces (PAC1 +). In der Kombination kann die erfindungsgemäße Anti-Sinn-RNA als solche oder in Form eines sie kodierenden Vektors vorliegen. In the combination of the anti-sense RNA of the invention can be present as such or in the form of a vector encoding it. Ebenso kann die (ds)RNAse als solche oder in Form eines sie kodierenden Vektors vorliegen. Similarly, the (ds) RNAse may be present as such or in the form of a vector encoding it. Ein solcher Vektor kann ein üblicher Expressionsvektor sein. Such a vector may be a conventional expression vector. 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. It may be favorable when the expression of the coding of the (ds) RNAse sequence under the control of a constitutive or inducible promoter, such as a tissue or tumor-specific promoter. 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. Further, it may be advantageous if the combination is that a vector is present, the encoding both the inventive anti-sense RNA, as well as for the (ds) RNAse. Hinsichtlich des Vektors wird auf vorstehende Aus führungen verwiesen. In terms of the vector is made to the above From guides.

Die Kombination aus einer erfindungsgemäßen Anti-Sinn-RNA und einer (ds)RNAse kann in Zellen eingebracht werden. The combination of an inventive anti-sense RNA and a (ds) RNAse can be introduced into cells. Hinsichtlich der Zellen und des Einbringens der Anti-Sinn-RNA wird auf vorstehende Ausführungen verwiesen. With respect to the cells and the introduction of anti-sense RNA, reference is made to the above statements. Die (ds)RNAse kann als solche, dh als Protein, durch übliche Verfahren, wie Lipofektion, eingebracht werden. The (ds) RNAse can be used as such, ie are introduced as a protein, by conventional methods, such as lipofection. In Form eines sie kodierenden Vektors kann die (ds)RNAse durch Ver fahren eingebracht werden, wie sie für die Anti-Sinn-RNA genannt wurden. In the form of a vector, the encoding them (ds) RNAse can be introduced through drive Ver, as they were known 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. The present invention provides an anti-sense RNA, and a combination containing them ready to effect a strong inhibition of gene expression. Die vorliegende Erfindung findet somit eine breite Anwendung in der Molekularbiologie und der Medizin. The present invention thus is widely used in molecular biology and medicine. Insbesondere kann an die Diagnose und/oder Therapie von Erkrankungen gedacht werden, bei denen einzelne Proteine auslösend oder ver stärkend sind. In particular, can be thought of diagnosis and / or treatment of diseases in which individual proteins are triggering or strengthening ver. Dies sind z. These are for. B. Erkrankungen, bei denen Hormone eine große Rolle spielen, Tumorerkrankungen und virale Infektionen, wie HIV und AIDS. As diseases in which hormones play a major role in tumor diseases and viral infections, such as HIV and AIDS.

Kurze Beschreibung der Zeichnung Brief Description of Drawing

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

Fig. 2 zeigt die Hemmung der Genexpression durch eine erfindungsgemäße Anti-Sinn-RNA. Fig. 2 shows the inhibition of gene expression by an inventive anti-sense 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. (1) the rate of expression of the CAT gene in applicatio ence of an anti-sense RNA with secondary structure I. (2) ex is pressionsrate 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 1 example 1 Herstellung von Expressions-Vektoren, die das Chloramphenicolace tyltransferase (CAT)-Gen in 5'→3' bzw. 3'→5' Richtung enthalten include the preparation of expression vectors tyltransferase the Chloramphenicolace (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), 1068) inseriert. The CAT gene was isolated from a conventional CAT-vector, and "multiple cloning site" in which the expression vector pJ3Ω (see FIG. Nucleic Acids Res. 18, (1990), 1068) inserted. In einem Fall erfolgte die Insertion in 5'→3' Richtung und es wurde der Expressionsvektor pJ3Ω-CAT erhalten. In one case the insertion took place in the 5 '→ 3' direction and the expression vector pJ3Ω-CAT was obtained. Im anderen Fall erfolgte die Insertion in 3'→5' Richtung und es wurde der Expressionsvektor pJ3Ω-TAC erhalten. In the other case, the insertion was '→ 5' direction in Figure 3 and the expression vector pJ3Ω-TAC was obtained.

Beispiel 2 example 2 Herstellung von Expressionsvektoren, die das CAT-Gen in 3'→5' Richtung und eine für eine Sekundärstruktur I bzw. II kodierende Sequenz enthalten Preparation of expression vectors containing the CAT gene in the 3 '→ 5' direction and a sequence coding for a secondary structure I or II sequence (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 -Sequenz 1. Preparation of a (GC) 20 -EcoRI- (GC) 20 sequence

  • (a) Mittels eines automatischen Synthese-Geräts (Oligonukleotid- Synthesizer) wurden 2 Oligodesoxynukleotide hergestellt: (A) using an automatic synthesis device (oligonucleotide synthesizer) were prepared 2 oligodeoxynucleotides:
  • (b) Die beiden Oligodesoxynukleotide wurden im Verhältnis 1 : 1 gemischt, auf 90°C erhitzt, danach langsam unter "annea ling"-Bedingungen auf Raumtemperatur abgekühlt. (B) The two oligodeoxynucleotides were in the ratio of 1: 1, heated to 90 ° C, then slowly cooled under "annea ling" conditions at room temperature. Dabei ent stand ein DNA Doppelstrang folgender Struktur: Here ent was a DNA double strand following structure:
  • (c) Unter Ligationsbedingungen entstanden Vielfache der in (b) beschriebenen DNA (C) ligation conditions created multiples of the DNA described in (b)
  • (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 separated by gel electrophoresis according to size, 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 inserted first into the EcoRI site of the usual cloning vector pBluescript (Stratagene), from which they are taken out by appropriate restriction enzymes for subcloning in-the vector comprising the CAT gene in the 3 '→ 5' direction could.
2. Einbau der (GC) 20 -EcoRI-(GC) 20) Sequenz in den Vektor, der das CAT- Gen in 3'→5' Richtung aufweist 2. Installation of the (GC) 20 -EcoRI- (GC) 20) sequence into the vector comprising 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. The vector pJ3Ω-TAC of Example 1 was "multiple cloning site" cut in between the promoter and the TAC insert with appropriate restriction enzymes. Die (GC) 20 -EcoRI-(GC) 20) Sequenz wurde mit den entsprechenden Enzymen aus dem pBluescript-Vektor von Beispiel 2(e) entnommen. (GC) 20 -EcoRI- (GC) 20) sequence was taken with the corresponding enzymes from the pBluescript vector of Example 2 (e). Die beiden Nukleinsäuren wurden per Ligation verbunden. The two nucleic acids were linked by ligation. Es wurde der Expressionsvektor pJ3Ω-TAC-Sek. It was the expression vector pJ3Ω TAC sec. I erhalten. I receive.

(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. The (GC) prepared in Example 2 (A) 20 -EcoRI- (GC) 20 sequence was inserted into the vector pJ3Ω-TAC at the 3'-end of the TAC gene. Es wurde der Expressionsvektor pJ3Ω-TAC-Sek.II erhalten. the expression vector pJ3Ω TAC Sek.II was obtained.

Beispiel 3 example 3 Herstellung eines Expressionsvektors, der für eine (ds) RNAse kodiert Preparing an expression vector encoding 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. From a conventional genomic library of the Schizosaccharomyces pombe gene (PAC1 +) coding for a (ds) RNAse was isolated by PCR amplification. Hierzu wurden Primer verwendet, die aus der bekannten Sequenz des Gens pac1+ (vgl. Datenbank: embl: S78982) abgeleitet worden waren. To this end primers were used that from the known sequence of the gene PAC1 + (cf. database: EMBL: S78982.) Were derived. Das Gen pac1+ wurde in dem bekannten Vektor pBluescript kloniert und durch Sequenzierung bestätigt. The gene was cloned PAC1 + in the well-known vector pBluescript and confirmed by sequencing. Nach Umklonierung in den üblichen Expressionsvektor pcDNA3 (InVitrogen) wurde der Expressionsvektor pcDNA3-pac1+ erhalten. After cloning in the usual expression vector pcDNA3 (InVitrogen) expression vector was obtained pcDNA3-PAC1 +.

Beispiel 4 example 4 Hemmung der Genexpression durch eine Anti-Sinn-RNA mit Sekun därstruktur Inhibition of gene expression by antisense RNA with seconding därstruktur

  • (a) Ehrlich Ascites Tumorzellen (10 7 Zellen/ml) wurden mit den Expressionsvektoren pJ3Ω-CAT, pJ3Ω-TAC, pJ3Ω-TAC-Sek. (a) Ehrlich ascites tumor cells (10 7 cells / ml) were incubated with the expression vectors pJ3Ω-CAT, pJ3Ω-TAC, TAC pJ3Ω sec. I bzw. pJ3Ω-TAC-Sek. I or pJ3Ω TAC sec. II transfiziert (vgl. Tabelle 1). transfected II (see. Table 1). Die Transfektion wurde mittels Elektroporation (366V/950yF/Ele ktrodenabstand D=4mm) durchgeführt. Transfection was by electroporation (366V ktrodenabstand / 950yF / Ele D = 4 mm) was performed. 24 h nach Transfek tion wurden die Zellen geerntet, lysiert und Aliquote mit radio aktiv markiertem Chloramphenicol inkubiert. 24 h after Transfek tion, the cells were harvested, lysed and aliquots incubated with radiolabeled chloramphenicol. Es wurde die Konversionsrate (in Ac-, Di-Ac-Chloramphenicol) nach DC durch Messung der Radioaktivität bestimmt. It has been the conversion rate (in Ac, di-Ac-chloramphenicol) to DC by measuring the radioactivity determined.

    Tabelle 1 Table 1

Aus Fig. 1 geht hervor, daß durch Transfektion von pJ3Ω-TAC-Sek. From Fig. 1 it is apparent that by transfection of pJ3Ω TAC sec. I bzw. pJ3Ω-TAC-Sek. I or pJ3Ω TAC sec. 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. II (Fig. See FIG. 1, (2), (3) a stronger inhibition of Expres sion of the CAT gene can be achieved than when pJ3Ω-TAC (see. Fig. 1 is used (1).

  • (b) Ehrlich Ascites Tumorzellen (10- 7 Zellen/ml) wurden mit den Expres sionsvektoren pJ3Ω-CAT, pJ3Ω-TAC-Sek. (b) Ehrlich ascites tumor cells (10 7 cells / ml) were incubated with the Expres sion vectors pJ3Ω-CAT, pJ3Ω TAC sec. I bzw. pcDNA3-pac1+ tranfiziert (vgl. Tabelle 2). I or pcDNA3-PAC1 + transfected (see. Table 2). Die Transfektionsbedingungen waren wie in Beispiel 4 (a) beschrieben. The transfection conditions were as described in Example 4 (a).

    Tabelle 2 table 2

Aus Fig. 2 geht hervor, daß durch Kotransfektion von pJ3Ω-TAC-Sek. From Fig. 2 it is apparent that by co-transfection of pJ3Ω TAC sec. I mit pcDNA3-pac1+ (vgl. Fig. 2 (2)) eine stärkere Hemmung der Expression von CAT erhalten wird, als wenn pJ3Ω-TAC-Sek. I with pcDNA3-PAC1 + ((see. Fig. 2: 2)) is obtained a stronger inhibition of the expression of CAT than if pJ3Ω TAC sec. I (vgl. Fig. 2, (1) alleine ver wendet wird. I (see. Fig. 2, (1) applies 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. Thus clear that an anti-sense RNA with secondary structure on gene expression has a greater inhibitory effect as an anti-sense RNA without secondary structure. 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. Further, the inhibitory effect of the anti-sense RNA may be further enhanced with secondary structure, if in addition to optionally naturally occurring (ds) RNAses a (ds) RNAse activity caused by means of the method described or generated becomes clear.

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, wherein the anti-of 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 seconding därstruktur at the 5'- and / or 3'-end of the anti-sense RNA has been created.
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 of a (GC) n -Palindrom- (GC) n - sequence - or (CG) n -Palindrom- (CG) n.
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. A combination comprising the antisense 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. A combination according to claim 5, characterized in that the (ds) RNAse present 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 antisense RNA according to any one of claims 1-4 and of the combination according to claim 5 or 6 for the inhibition of gene expression.
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