EP0918853A2 - Antisense rna with a secondary structure - Google Patents

Antisense rna with a secondary structure

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Publication number
EP0918853A2
EP0918853A2 EP97936610A EP97936610A EP0918853A2 EP 0918853 A2 EP0918853 A2 EP 0918853A2 EP 97936610 A EP97936610 A EP 97936610A EP 97936610 A EP97936610 A EP 97936610A EP 0918853 A2 EP0918853 A2 EP 0918853A2
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EP
European Patent Office
Prior art keywords
sense rna
expression
vector
secondary structure
pj3ω
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP97936610A
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German (de)
French (fr)
Inventor
Dieter Werner
Christof Granzow
Gaby Joswig
Karsten Rothbarth
Marie Schubert
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Deutsches Krebsforschungszentrum DKFZ
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Deutsches Krebsforschungszentrum DKFZ
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Publication of EP0918853A2 publication Critical patent/EP0918853A2/en
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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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention relates to an anti-sense RNA with a secondary structure, a combination containing it and the use of both.
  • 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.
  • 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 encoding it. Such a vector can be a common expression vector. It can 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 promoter.
  • 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, these can be the same or different from one another.
  • Complicated palindromes such as (AGCT) n or (GAATTC) n are also preferred.
  • An anti-sense RNA according to the invention can be produced by customary methods. It is favorable to produce 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 the 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.
  • GC double-stranded
  • EcoRI GC
  • An anti-sense RNA according to the invention can be introduced into cells as such or in the form of a vector encoding it.
  • the cells can be any cells, such as plant and animal, especially mammalian and very particularly human cells.
  • the cells can be inside or outside of an organism. The latter can be freshly isolated or kept in culture.
  • the anti-sense RNA can be introduced into the cells by conventional transfection techniques, such as electroporation.
  • Another object of the present invention is a combination of an 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, for example, in the yeast strain Schizosaccharomyces pombe (pad +).
  • the anti-sense RNA according to the invention can be present 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.
  • 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
  • the combination consists in the presence of a vector which codes both for the anti-sense RNA according to the invention and for the (ds) RNAse.
  • vector With regard to the vector, reference is made to the above statements.
  • the combination of an anti-sense RNA according to the invention and a (ds) RNAse can be introduced into cells.
  • the (ds) RNAse as such, i.e. as a protein, by conventional methods such as lipofection.
  • the form of a vector encoding it the
  • RNAse can be introduced by methods as they were called for the anti-sense RNA.
  • the present invention provides an anti-sense RNA and a combination containing it which cause potent inhibition of gene expression.
  • the present invention is thus widely used in molecular biology and medicine.
  • diseases in which individual proteins trigger or reinforce are, for example, diseases in which hormones play a major role, tumor diseases and viral infections, such as HIV and AIDS.
  • diseases in which hormones play a major role are, for example, diseases in which hormones play a major role, tumor diseases and viral infections, such as HIV and AIDS.
  • FIG. 1 shows the inhibition of gene expression by an anti-sense RNA according to the invention.
  • (1) is the rate of expression of the CAT gene in the presence of an anti-sense RNA.
  • (2) is the expression rate of the
  • CAT gene in the presence of an anti-sense RNA with secondary structure I.
  • (3) is the expression rate of the CAT gene in the presence of an anti-sense RNA with secondary structure II.
  • FIG. 2 shows the inhibition of gene expression by an anti-sense RNA according to the invention.
  • (1) is the rate of expression of the CAT gene in the presence of an anti-sense RNA with secondary structure I.
  • (2) is the rate of expression of the CAT gene in the presence of an anti-sense RNA with secondary structure I and a (ds) RNAse.
  • Example 1 Production of expression vectors which contain the chioramphenicolacetyl transferase (CAT) gene in the 5 ' ⁇ 3' or 3 ' ⁇ 5' direction.
  • CAT chioramphenicolacetyl transferase
  • the CAT gene was isolated from a conventional CAT vector and inserted into the "multiple cloning site" of the expression vector pJ3 ⁇ (cf. Nuclear acids res. 1 8, (1 990), 1068). In one case 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 Production of expression vectors which contain the CAT gene in the 3 ' ⁇ 5' direction and a sequence coding for a secondary structure I or II.
  • AATTC- (GO 20-GAATTC- (GC) 20-GAATTC- (GC) 0-G.
  • Cloning vector pBluescript (Stratagene) was used, from which it could be removed by suitable restriction enzymes for recloning into the vector which has the CAT gene in the 3 '- * 5' direction.
  • the vector pJ3 ⁇ -TAC from Example 1 was cut in the "multiple cloning site" between the promoter and the TAC insert with suitable restriction enzymes.
  • Sequence was taken from the pBluescript vector of Example 2 (e) with the appropriate enzymes. The two nucleic acids were linked by ligation.
  • Example 3 Preparation of an expression vector which codes for a (ds) RNAse.
  • the gene (pad +) coding for a (ds) RNAse was isolated from a conventional genomic library of Schizosaccharomyces pombe by means of PCR amplification. For this purpose, primers were used which had been derived from the known sequence of the pad + gene (cf. database: embl: S78982). 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.
  • Example 4 Inhibition of gene expression by an anti-sense RNA with a secondary structure
  • Ehrlich ascites tumor cells (10 7 cells / ml) were expressed with the expression vectors pJ3 ⁇ -CAT, pJ3 ⁇ -TAC, pJ3 ⁇ -TAC-sec. I or pJ3 ⁇ -TAC-sec. II transfected (see Table 1).
  • RNAse activity is produced or generated by means of the described methods.

Abstract

An antisense RNA with special secondary structures is disclosed, as well as a combination of the antisense RNA and of a (ds)RNAse. The antisense RNA and its combination may be used to inhibit gene expression.

Description

Anti-Sinn-RNA mit Sekundärstruktur Anti-sense RNA with secondary structure
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 it and the use of both.
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 eine Anti-Sinn-RNA mit besonderen Sekundärstrukturen erreicht.According to the invention, this is achieved by an anti-sense RNA with special secondary structures.
Mit dem Ausdruck "besonderer Sekundärdruck" ist gemeint, daß es sich nicht um eine natürlich vorkommende Sekundärstruktur handelt, sondern daß diese künstlich erzeugt worden ist.The expression "special secondary pressure" means 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, 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 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-spezifischen 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 encoding it. Such a vector can be a common expression vector. It can 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 promoter.
Der Ausdruck "Sekundärstruktur" umfaßt jegliche DNA- und/oder RNA-Sequenz, die in einer Anti-Sinn-RNA vorliegen kann und eine zumindest teilweise "Hair- pin"-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 Sekundärstrukturen vor, können diese gleich oder verschieden voneinander sein. Vorzugsweise 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, these can be the same or different from one another. The secondary structure is preferably a (GC) n -palindrome (GC) n -, <AT) n -palindrome (AT) n -, or (CG) n -palindrome (CG) n sequence, with particular preference is when 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 Labora- tory Press, 1 989, verwiesen.An anti-sense RNA according to the invention can be produced by customary methods. It is favorable to produce 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 the 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, 1 989.
Eine erfindungsgemäße Anti-Sinn-RNA kann als solche oder 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. 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 (pad + ) . 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 konstituti- ven oder induzierbaren Promotors, wie eines Gewebe- oder Tumor-spezifischenAn anti-sense RNA according to the invention can be introduced into cells as such or in the form of a vector encoding it. The cells can be any cells, such as plant and animal, especially mammalian and very particularly human cells. The cells can be inside or outside of an organism. The latter can be freshly isolated or kept in culture. The anti-sense RNA can be introduced into the cells by conventional transfection techniques, such as electroporation. Another object of the present invention is a combination of an 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, for example, in the yeast strain Schizosaccharomyces pombe (pad +). In combination, the anti-sense RNA according to the invention can be present 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. 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. 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 Ausführungen verwiesen.Promoter, stands. Furthermore, it may be advantageous if the combination consists in the presence of a vector which codes both for the anti-sense RNA according to the invention and for the (ds) RNAse. With regard to the vector, reference is made to the above statements.
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 dieThe combination of an anti-sense RNA according to the invention and a (ds) RNAse can be introduced into cells. With regard to the cells and the introduction of the anti-sense RNA, reference is made to the above statements. The (ds) RNAse as such, i.e. as a protein, by conventional methods such as lipofection. In the form of a vector encoding it, the
(ds)RNAse durch Verfahren eingebracht werden, wie sie für die Anti-Sinn-RNA genannt wurden.(ds) RNAse can be introduced by methods 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 verstärkend sind. Dies sind z.B. Erkrankungen, bei denen Hormone eine große Rolle spielen, Tumorerkrankungen und virale Infektionen, wie HIV und AIDS. Kurze Beschreibung der ZeichnungThe present invention provides an anti-sense RNA and a combination containing it which cause potent inhibition of gene expression. The present invention is thus widely used in molecular biology and 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, such as HIV and AIDS. Brief 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 Anwesenheit einer Anti-Sinn-RNA. (2) ist die Expressionsrate des1 shows the inhibition of gene expression by an anti-sense RNA according to the invention. (1) is the rate of expression of the CAT gene in the presence of an anti-sense RNA. (2) is the expression rate of the
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.CAT gene in the presence of an anti-sense RNA with secondary structure I. (3) is the expression rate 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 Anwesenheit einer Anti-Sinn-RNA mit Sekundärstruktur I. (2) ist die Expressionsrate des CAT-Gens in Anwesenheit einer Anti-Sinn-RNA mit Sekundärstruktur I und einer (ds)RNAse.2 shows the inhibition of gene expression by an anti-sense RNA according to the invention. (1) is the rate of expression of the CAT gene in the presence of an anti-sense RNA with secondary structure I. (2) is the rate of expression 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 : Herstellung von Expressions- Vektoren, die das Chioramphenicolace- tyltransferase (CAT)-Gen in 5'→ 3' bzw. 3'→ 5' Richtung enthalten.Example 1: Production of expression vectors which contain the chioramphenicolacetyl 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. Nu- cleic acids res. 1 8, ( 1 990), 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. Beispiel 2: Herstellung von Expressionsvektoren, die das CAT-Gen in 3'→ 5' Richtung und eine für eine Sekundärstruktur I bzw. II kodierende Sequenz enthalten.The CAT gene was isolated from a conventional CAT vector and inserted into the "multiple cloning site" of the expression vector pJ3Ω (cf. Nuclear acids res. 1 8, (1 990), 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. Example 2: Production of expression vectors which contain the CAT gene in the 3 '→ 5' direction and a sequence coding for a secondary structure I or II.
(A) Expressionsvektor mit einer (GC)20-EcoRI-{GC)20-Sequeπz 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-(a) Using an automatic synthesis device (oligonucleotide
Synthesizer) wurden 2 Oligodesoxynukleotide hergestellt:2 oligodeoxynucleotides were produced:
AATTC- (GC) 20-G undAATTC- (GC) 20-G and
G- (GC ) 2θ-CTTAAG- (GC) 2 θ-CTTAA
(b) Die beiden Oligodesoxynukleotide wurden im Verhältnis 1 : 1 gemischt, auf 90°C erhitzt, danach langsam unter "annea- Iing"-Bedingungen auf Raumtemperatur abgekühlt. Dabei ent- stand ein DNA Dopplestrang 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 of the following structure:
AATTC- (GC ) 20-G * * * *AATTC- (GC) 20-G * * * *
G- ( GC ) 20-CTTAAG- (GC) 20-CTTAA
(c) Unter Ligationsbedingungen entstanden Vielfache der in (b) beschriebenen DNA(c) Multiples of the DNA described in (b) were generated under ligation conditions
AATTC- (GO 20-GAATTC- (GC ) 20-GAATTC- (GC ) 0-G .AATTC- (GO 20-GAATTC- (GC) 20-GAATTC- (GC) 0-G.
* ** ****** ** ***** ** →,* ** ****** ** ***** ** →,
G- (GC) 20-CTTAAG- (GC) 20-CTTAAG- (GC) 20-CTTAA..G- ( GC ) 20-CTTAAG- (GC) 20-CTTAAG- (GC) 20-CTTAA ..
(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 refined by gel electrophoresis Size separated and a sequence consisting of dimers was eluted from the gel and phosphorylated using polynucleotide kinase / ATP.
AATTC- (GC) 0-GAATTC- (GC) 20-G-PAATTC- (GC) 0-GAATTC- (GC) 20-G-P
* * * * * * * * * * * ** * * * * * * * * * * *
P-G- ( GC ) 20-CTTAAG- (GC) 20"CPG- (GC) 20-CTTAAG- (GC) 2 0 "C
(e) Diese Sequenz wurde zunächst in die EcoRI-Stelle des üblichen(e) This sequence was first inserted into the EcoRI site of the usual
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.Cloning vector pBluescript (Stratagene) was used, from which it could be removed by suitable restriction enzymes for recloning into the vector which has the CAT gene in the 3 '- * 5' direction.
2. Einbau der {GC)2o-EcoRI-(GC)2o) Sequenz in den Vektor, der das CAT- Gen in 3'→ 5' Richtung aufweist.2. Incorporation of the {GC) 2 o-EcoRI (GC) 2 o) 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 was taken from the pBluescript vector of Example 2 (e) with the appropriate enzymes. 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.ll erhalten. Beispiel 3: Herstellung eines Expressionsvektors, der für eine (ds) RNAse kodiert.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-Sek.ll was obtained. Example 3: Preparation of an expression vector which codes for a (ds) RNAse.
Aus einer üblichen genomischen Bibliothek von Schizosaccharomy- ces pombe wurde mittels einer PCR-Amplifikation das für eine (ds)RNAse kodierende Gen (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.The gene (pad +) coding for a (ds) RNAse was isolated from a conventional genomic library of Schizosaccharomyces pombe by means of PCR amplification. For this purpose, primers were used which had been derived from the known sequence of the pad + gene (cf. database: embl: S78982). 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 4: Hemmung der Genexpression durch eine Anti-Sinn-RNA mit SekundärstrukturExample 4: Inhibition of gene expression by an anti-sense RNA with a secondary 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/950μF/Ele- ktrodenabstand 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. (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 / 950μF / 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.
Tabelle 1 :Table 1 :
pJ3Ω-CAT 3//g 3/yg 3/ gpJ3Ω-CAT 3 // g 3 / yg 3 / g
pJ3Ω-TAC-Sek. I - 7,5/ygpJ3Ω TAC sec. I - 7.5 / yg
pJ3Ω-TAC-Sek. II - - 7,5μgpJ3Ω TAC sec. II - - 7.5μg
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 Expression des CAT-Gens erreicht werden kann, als wenn ρJ3Ω-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 ρJ3Ω-TAC (cf. FIG. 1, (1) is used.
(b) Ehrlich Ascites Tumorzellen ( 107 Zellen/ml) wurden mit den Expressionsvektoren pJ3Ω-CAT, pJ3Ω-TAC-Sek. I bzw. pcDNA3-pad + tranfiziert (vgl. Tabelle 2). Die Transfektionsbedingungen waren wie in Beispiel 4 (a) beschrieben.(b) Ehrlich ascites tumor cells (10 7 cells / ml) were expressed with the expression vectors pJ3Ω-CAT, pJ3Ω-TAC-Sek. I or pcDNA3-pad + transfected (see Table 2). The transfection conditions were as described in Example 4 (a).
Tabelle 2:Table 2:
1 2 pJ3Ω-CAT 5 vg 5μg pJ3Ω-TAC-Sek. 10/yg 10/yg pcDNA3-pad + - 10/yg1 2 pJ3Ω-CAT 5 vg 5μg pJ3Ω-TAC-sec. 10 / yg 10 / yg pcDNA3-pad + - 10 / yg
Aus Fig. 2 geht hervor, daß durch Kotransfektion von pJ3Ω-TAC-Sek. I mit pcDNA3-pad + (vgl. Fig. 2 (2)) eine stärkere Hemmung der Expres- sion von CAT erhalten wird, als wenn pJ3Ω-TAC-Sek. I (vgl. Fig. 2, ( 1 ) alleine verwendet wird. Somit wird deutlich, 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 eineFrom Fig. 2 it appears that by co-transfection of pJ3Ω-TAC-sec. I with pcDNA3-pad + (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. It is thus clear that an anti-sense RNA with a secondary structure has a greater inhibitory effect on gene expression than an anti-sense RNA without a secondary structure. It is also clear that the inhibitory effect of the anti-sense RNA with a secondary structure can be increased if, in addition to any naturally present (ds) RNAses
(ds)RNAse-Aktivität mittels der beschriebenen Verfahren hervorgerufen bzw. erzeugt wird. (ds) RNAse activity is produced or generated by means of the described methods.

Claims

Patentansprüche claims
1 . Anti-Sinn-RNA mit besonderen Sekundärstrukturen.1 . Anti-sense RNA with special secondary structures.
2. Anti-Sinn-RNA nach Anspruch 1 , dadurch gekennzeichnet, daß die Sekundärstruktur am 5'- und/oder 3'-Ende der Anti-Sinn-RNA geschaffen wor- den ist.2. Anti-sense RNA according to claim 1, characterized in that the secondary structure 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 (GOn-PalindrorrHGC),,- 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 (GO n -PalindrorrHGC) ,, - 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. Anti-Sinn-RNA nach einem der Ansprüche 1 - 4, dadurch gekennzeichnet, daß die Anti-Sinn-RNA durch einen Vektor kodiert ist.5. Anti-sense RNA according to one of claims 1-4, characterized in that the anti-sense RNA is encoded by a vector.
6. Kombination, umfassend die Anti-Sinn-RNA nach einem der Ansprüche 1 - 5 und eine (ds)RNAse.6. Combination comprising the anti-sense RNA according to any one of claims 1-5 and a (ds) RNAse.
7. Kombination nach Anspruch 6, dadurch gekennzeichnet, daß die Anti-7. Combination according to claim 6, characterized in that the anti
Sinn-RNA und die (ds)RNAse durch einen oder mehrere Vektoren kodiert sind.Sense RNA and the (ds) RNAse are encoded by one or more vectors.
8. Verwendung der Anti-Sinn-RNA nach einem der Ansprüche 1 -5 und der Kombination nach Anspruch 6 oder 7 zur Hemmung der Genexpression. 8. Use of the anti-sense RNA according to one of claims 1 -5 and the combination according to claim 6 or 7 for inhibiting gene expression.
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