WO1998005770A2

WO1998005770A2 - Antisense rna with a secondary structure

Info

Publication number: WO1998005770A2
Application number: PCT/DE1997/001691
Authority: WO
Inventors: Dieter Werner; Christof Granzow; Gaby Joswig; Karsten Rothbarth; Marie Schubert
Original assignee: Deutches Krebsforschungszentrum Stiftung Des Öffentlichen Rechts
Priority date: 1996-08-07
Filing date: 1997-08-05
Publication date: 1998-02-12
Also published as: EP0918853A2; WO1998005770A3; DE19631919A1; DE19631919C2

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-sense RNA with secondary structure

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.

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.

The present invention is therefore based on the object of providing a means with which a strong inhibition of gene expression can be achieved.

According to the invention, this is achieved by an anti-sense RNA with special secondary structures.

The expression "special secondary pressure" means that it is not a naturally occurring secondary structure, but that it has been created artificially.

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.

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.

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.

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 +). 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

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.

The 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 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. 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

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.

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.

The invention is illustrated by the following examples.

Example 1: Production of expression vectors which contain the chioramphenicolacetyl transferase (CAT) gene in the 5 '→ 3' or 3 '→ 5' direction.

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) Expression vector with a (GC) ₂₀ -EcoRI- (GC) ₂₀ sequence at the 5 'end of the CAT gene (secondary structure I)

1 . Preparation of a (GC) ₂₀ EcoRI (GC) ₂₀ sequence.

(a) Using an automatic synthesis device (oligonucleotide

2 oligodeoxynucleotides were produced:

AATTC- (GC) 20-G and

G- (GC) ₂ θ-CTTAA

(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 * * * *

G- (GC) 20-CTTAA

(c) Multiples of the DNA described in (b) were generated under ligation conditions

AATTC- (GO 20-GAATTC- (GC) 20-GAATTC- (GC) 0-G.

* ** ****** ** ***** ** →,

G- ⁽ GC ⁾ 20-CTTAAG- (GC) 20-CTTAAG- (GC) 20-CTTAA ..

(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-P

* * * * * * * * * * * *

PG- (GC) 20-CTTAAG- (GC) ₂ 0 "C

(e) This sequence was first inserted into the EcoRI site of the usual

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. Incorporation of the {GC) ₂ o-EcoRI (GC) ₂ o) sequence into the vector which has the CAT gene in the 3 '→ 5' direction.

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) Expression vector with a (GC) ₂₀ EcoRI (GC, ₂₀ sequence at the 3 'end of the CAT gene (secondary structure II).

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.

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

(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.

Table 1 :

pJ3Ω-CAT 3 // g 3 / yg 3 / g

pJ3Ω TAC sec. I - 7.5 / yg

pJ3Ω TAC sec. II - - 7.5μg

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 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).

Table 2:

1 2 pJ3Ω-CAT 5 vg 5μg pJ3Ω-TAC-sec. 10 / yg 10 / yg pcDNA3-pad + - 10 / yg

From 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 activity is produced or generated by means of the described methods.

Claims

claims

1 . Anti-sense RNA with special secondary structures.

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-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-sense RNA according to claim 3, characterized in that n = 20 and the palindrome is an EcoRI restriction site.

5. Anti-sense RNA according to one of claims 1-4, characterized in that the anti-sense RNA is encoded by a vector.

6. Combination comprising the anti-sense RNA according to any one of claims 1-5 and a (ds) RNAse.

7. Combination according to claim 6, characterized in that the anti

Sense RNA and the (ds) RNAse are encoded by one or more vectors.

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.