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WO1998005770A2 - Arn antisens avec une structure secondaire - Google Patents

Arn antisens avec une structure secondaire Download PDF

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Publication number
WO1998005770A2
WO1998005770A2 PCT/DE1997/001691 DE9701691W WO9805770A2 WO 1998005770 A2 WO1998005770 A2 WO 1998005770A2 DE 9701691 W DE9701691 W DE 9701691W WO 9805770 A2 WO9805770 A2 WO 9805770A2
Authority
WO
WIPO (PCT)
Prior art keywords
sense rna
expression
vector
secondary structure
pj3ω
Prior art date
Application number
PCT/DE1997/001691
Other languages
German (de)
English (en)
Other versions
WO1998005770A3 (fr
Inventor
Dieter Werner
Christof Granzow
Gaby Joswig
Karsten Rothbarth
Marie Schubert
Original Assignee
Deutches Krebsforschungszentrum Stiftung Des Öffentlichen Rechts
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Deutches Krebsforschungszentrum Stiftung Des Öffentlichen Rechts filed Critical Deutches Krebsforschungszentrum Stiftung Des Öffentlichen Rechts
Priority to EP97936610A priority Critical patent/EP0918853A2/fr
Publication of WO1998005770A2 publication Critical patent/WO1998005770A2/fr
Publication of WO1998005770A3 publication Critical patent/WO1998005770A3/fr

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Classifications

    • 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.

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

Abstract

L'invention concerne un ARN antisens avec des structures secondaires particulières, ainsi qu'une combinaison de cet ARN antisens et d'une (ds)RNase. L'ARN antisens et cette combinaison sont utiles pour inhiber l'expression de gènes.
PCT/DE1997/001691 1996-08-07 1997-08-05 Arn antisens avec une structure secondaire WO1998005770A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP97936610A EP0918853A2 (fr) 1996-08-07 1997-08-05 Arn antisens avec une structure secondaire

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19631919.6 1996-08-07
DE1996131919 DE19631919C2 (de) 1996-08-07 1996-08-07 Anti-Sinn-RNA mit Sekundärstruktur

Publications (2)

Publication Number Publication Date
WO1998005770A2 true WO1998005770A2 (fr) 1998-02-12
WO1998005770A3 WO1998005770A3 (fr) 1998-03-26

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PCT/DE1997/001691 WO1998005770A2 (fr) 1996-08-07 1997-08-05 Arn antisens avec une structure secondaire

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EP (1) EP0918853A2 (fr)
DE (1) DE19631919C2 (fr)
WO (1) WO1998005770A2 (fr)

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DE19631919A1 (de) 1998-02-12
WO1998005770A3 (fr) 1998-03-26
EP0918853A2 (fr) 1999-06-02
DE19631919C2 (de) 1998-07-16

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