WO2003095652A2

WO2003095652A2 - Expression constructs for producing double-stranded (ds) rna and the use thereof

Info

Publication number: WO2003095652A2
Application number: PCT/EP2003/004835
Authority: WO
Inventors: Wolfgang Liebetrau; Dieter Link
Original assignee: Xantos Biomedicine Ag
Priority date: 2002-05-08
Filing date: 2003-05-08
Publication date: 2003-11-20
Also published as: WO2003095652A3; AU2003239851A1

Abstract

The invention relates to a polynucleotide containing an inner polynucleotide, which is functionally linked to a first eukaryotic expression control sequence at the 5' end and which is functionally linked to a second eukaryotic expression control sequence at the 3' end, whereby: (i) only the first eukaryotic expression control sequence is functionally linked to a first polyadenylation sequence at the 5' end, and the polyadenylation sequence is functional in the 3' to 5' orientation or; (ii) only the second eukaryotic expression control sequence is functionally linked to a second polyadenylation sequence at the 3' end, and the polyadenylation sequence is functional in the 5' to 3' orientation or; (iii) the first eukaryotic expression control sequence is functionally linked to a first polyadenylation sequence at the 5' end and the polyadenylation sequence is functional in the 3' to 5' orientation, and the second eukaryotic expression control sequence is functionally linked to a second polyadenylation sequence at the 3' end and the polyadenylation sequence is functional in the 5' to 3' orientation. The invention also relates to methods for producing double-stranded polynucleotides. The invention additionally concerns vectors and mixtures of vectors, to methods for producing vectors that comprise the inventive polynucleotides or comprise the polynucleotides produced according to the inventive methods, and to host cells containing these vectors. Additionally disclosed are methods for identifying genes whose inactivation leads to detectable changes in a target cell. The invention additionally relates to transgenic animals containing an inventive polynucleotide. Finally, the invention discloses the use of the inventive polynucleotide for producing a medicament for treating and preventing diseases.

Description

Expression constructs for the production of double-stranded (ds) RNA and their

application

The invention relates to a polynucleotide containing an inner polynucleotide which is operatively linked at the 5 ^' end to a first eukaryotic expression control sequence and at the 3 ^' end is operably linked to a second eukaryotic expression control sequence, with (i) only the first eukaryotic expression control sequence at the 5 'end is functionally linked to a first polyadenylation sequence and the polyadenylation sequence is functional in 3 ^' after 5 ^' orientation, or (ii) only the second eukaryotic expression control sequence at the 3 'end is functionally linked with a second polyadenylation sequence and the polyadenylation sequence in 5 ^' after 3 ^' orientation is functional, or (iii) the first eukaryotic expression control sequence at the 5 'end is functionally linked to a first polyadenylation sequence and the polyadenylation sequence is functional in 3' after 5 ^' orientation and the second eukaryotic expression control sequence in turn is functionally linked at the 3 ^' end to a second polyadenylation sequence and the polyadenylation sequence is functional in 5 ^' after 3 'orientation. The invention further relates to methods for producing double-stranded polynucleotides. The invention also relates to vectors and mixtures of vectors and to methods for producing vectors which comprise the polynucleotides according to the invention or the polynucleotides produced by the methods of the invention, and to host cells which contain these vectors. Furthermore, the invention relates to methods for identifying genes, the inactivation of which leads to detectable changes in a target cell. The invention also relates to transgenic animals which contain a polynucleotide according to the invention. Finally, the invention relates to the use of the polynucleotide according to the invention for the manufacture of a medicament for the treatment and prevention of diseases. RNA interference (RNAi) describes the specific interaction of a nucleic acid with a sequence-homologous mRNA and the resulting reduction in gene expression in the literature. RNAi is a form of post-transcriptional gene silencing, a natural process that involves the inactivation of genes by double-stranded RNA (dsRNA). DsRNA is broken down into small fragments within the cells by specific enzymes. These RNA fragments activate one RNAi-induced enzyme complex (RISC), which destroys the mRNA which is complementary to the RNA fragments from the dsRNA, the activity of a specific gene is reduced or abolished by the destruction of the mRNA to "antisense" effects, RNA interference (RNAi) means the nucleolytic cleavage, guided by a double-stranded RNA (dsRNA), of one of the dsRNA sequence homologous mRNAs (Fire et al., 1998). The specific protein complex required for this must be activated by a dsRNA (Bass, 2000; Carthew, 2001). Accordingly, RNAi is induced by dsRNA, which originates from transgenes, transposons, viruses, or artificially introduced dsRNA. RNAi has been described in various species (Bosher and Labouesse, 2000). The best studied organisms are Arabidopsis thaliana, Caenorhabditis elegans, and Drosophila melanogaster. Furthermore, RNAi has been shown in Xenopus (Nakano et al., 2000), Hydra (Lohmann et al., 1999; Lohmann and Bosch, 2000) and Trypanosoma brucei (Shi et al., 2000). In addition, RNAi is also known in plant genetics under the name PTGS (“post-transcriptional gene silencing”). In addition to the degradation of an mRNA, a modulation of chromatin activity such as methylation of regions with homologous sequences is also here (Jones et al., 1999; Mette et al., 2000 ; Matzke et al., 2001) and a spreading of the dsRNA-dependent signal across the cell boundaries "spreading" (Voinnet et al., 1998; Voinnet et al., 2000; Matzke et al., 2001). In addition, a role of RNA-dependent RNA polymerases (RdRP) in the amplification of the inducing dsRNA has been described (Matzke et al., 2001; Dalmay et al., 2000). Provide studies on the necessary structure of the dsRNA molecule (Hutvagner et al., 2000) and (Thomas et al., 2001). Interference with mRNA, "chromatin silencing" (Kelly and Fire, 1998) and "spreading" (Tabara et al., 1998) of the dsRNA signal also function as specific dsRNA effects in C. elegans (Harbinder et al., 1997; Fire et al., 1998; Montgomery et al., 1998; Tabara et al., 1998; Fire, 1999; Bosher et al., 1999; Sharp, 1999; Grishok et al., 2000; Bosher and

Labouesse, 2000; Tavernarakis et al., 2000). In C. elegans, the analysis of RNAi

Mutants contributed significantly to elucidating the mechanism ((Tabara et al.,

1999; Hunter, 1999; Hunter, 2000).

In Drosophila, RNAi was found in embryos (Yang et al., 2000; Kennerdell and Carthew,

2000), in S2 / Schneider cells (Caplen et al., 2000; Clemens et al., 2000) and in their extract (Tuschl et al., 1999; Zamore et al., 2000; Elbashir et al., 2001) documented.

However, recent publications have shown that RNAi is also effective in mammalian cells. Elbashir et al. (2001), for example, used 21-nucleotide siRNA (small interfering RNAs) duplexes to make expression more endogenous and heterologous

Genes in various mammalian cell lines, such as 293 cells and HeLa

Cells to prevent. Paddison et al. (2002) have shown that long double-stranded RNAs (approximately 500 nucleotides) specifically express gene expression in

Mammalian cells such as P19 mouse embryonic carcinoma cells and C2 / C12 mouse

Myoblast cells. Furthermore, Brummelkamp et al. (2002) developed a system for the stable expression of "short interfering" (si) RNAs in mammalian cells.

However, the function of RNAi in mammalian cells is apparently developmentally limited and no longer detectable in later embryonic stages (Wianny and Zemicka-Goetz, 2000). Experiments conducted in mammalian cells suggest that there are at least two different ones

Answers to dsRNA, a non-specific as well as a specific dsRNA

Answer that compete for the dsRNA. Nonspecific RNAi effects are due, among other things, to the presence of an antiviral common in mammalian cells

Mechanism attributed, which is also referred to as interferon response.

Longer dsRNA molecules are inducers of the unspecific dsRNA response, provided that they are at least 30 base pairs long. Cellular proteins sense the dsRNA and initiate a general inhibition of cellular translation (Terenzi et al., 1999; Williams, 1999). This leads to an unspecific reduction of

Gene expression. The dsRNA activates two enzymes: PKR, which in its active form phosphorylates the translation initiation factor elF2a, which leads to a shutdown of protein synthesis, and 2 ', 5'-oligoadenylate synthetase, which forms a molecule that activates RNase L, a non- specific enzyme that degrades mRNAs (Elbashir et al, 2001). In the sequence-specific dsRNA response, RNA interference (RNAi), the initiating double-stranded RNA is first broken down into short interfering RNAs (siRNAs). The siRNAs (presumably) provide that

Sequence information that allows a specific degradation of a specific mRNA.

The actual lead molecules for the RNAi enzyme complex are 21-23-mer dsRNA molecules, which arise from processing from longer precursors. In cell-free Drosophila extracts it could be shown that the direct addition of

21-23 mer dsRNA does not lead to a comparable or no interference, in contrast to the use of longer dsRNA inducers which are processed to 21-23 mer (Elbashir et al., 2001). Shorter dsRNA molecules are then processed very slowly to 21-23 mer dsRNA. This speaks for the use of longer dsRNA molecules in Drosophila cell culture in order to achieve strong RNAi effects. However, chemically synthesized 21-mer dsRNA molecules can also be used

Achieve RNAi effects. The effectiveness of such a single dsRNA

Oligonucleotide may be highly position dependent, apparently due to the different accessibility of the target mRNA. When processing different dsRNA-21mers from a longer precursor, this problem obviously does not occur due to the availability of different dsRNA-21mers.

DsRNA for experimental purposes is usually used in an in vitro

Transcription system established (Hunter, 1999). Procaryotic T7

(or also T3) RNA polymerase binding sites attached to gene-specific primers and used in a gene-specific PCR reaction. The DNA templates of both strands obtained in this way are then used in one or in separate in vitro transcription reactions. The complementary RNA strands obtained therefrom individually or in one batch can be purified, optionally hybridized and then in various ways, e.g. by calcium-phosphate transfection (Ui-Tei et al., 2000), lipofection (Lin et al.,

2001) or microinjection (Tabara et al., 1998) into a target cell or

Target organism are introduced. In other cases, short dsRNA is also chemically synthesized (Elbashir et al., 2001). For C. elegans is a special one

Technique has been described that is based on the feeding of genetically manipulated

E. coli bacteria based on a prokaryotic dsRNA

Expression plasmid were transformed with oppositely arranged promoters

(Tabara et al., 1998; Timmons and Fire, 1998). In this case both in E. coli

RNA strands are made and the hybridized dsRNA is in the digestive tract of C. elegans added. Obviously the ds-RNA dependent can be found there

Spread the "silencing" signal over the entire organism (Tabara et al., 1998; Kamath et al., 2000).

The techniques described in the prior art for examining RNAi effects in eukaryotic host cells, in particular in mammalian cells, all provide that RNAi molecules must first be produced in vitro before they are introduced into the cells. Such techniques are time-consuming and not particularly effective due to numerous process and purification steps and the introduction of the RNA into the cells. Nevertheless, it would be very desirable to use RNAi effects in eukaryotic cells as well. In particular, therapeutically or diagnostically relevant target genes could thereby be identified and / or provided. Diseases that are based on a malfunction of such target genes could then also be treated with RNAi or their occurrence could be prevented by preventive measures.

The technical problem of the present invention is therefore to provide measures and methods which allow an effective and time-optimized use of the RNAi effect, in particular in eukaryotic host cells. This enables the diagnosis and therapy of diseases that are based on a malfunction of target genes of the RNAi effect.

The technical problem is solved by the embodiments described in the claims.

Thus, the present invention initially relates to a polynucleotide containing an inner polynucleotide which is functionally linked at the 5 ^' end to a first eukaryotic expression control sequence and at the 3' end is functionally linked to a second eukaryotic expression control sequence, wherein

(i) only the first eukaryotic expression control sequence at the 5 'end is functionally linked to a first polyadenylation sequence and the polyadenylation sequence is functional in 3' after 5 ^' orientation, or (ii) only the second eukaryotic

Expression control sequence at the 3 'end with a second

Polyadenylation sequence is functionally linked and the

Polyadenylation sequence in 5 'after 3 ^' orientation is functional, or

(iii) the first eukaryotic expression control sequence at the 5 'end is functionally linked to a first polyadenylation sequence and the polyadenylation sequence is functional in 3 ^' after 5 ^' orientation and the second eukaryotic expression control sequence in turn is functionally linked at the 3 ^' end to a second polyadenylation sequence and the polyadenylation sequence in 5 ^' to 3 ^'

Orientation is functional.

The term “polynucleotide” refers to a polymeric form of nucleotides of any length. However, the polynucleotides according to the invention must comprise at least the sequences mentioned above. They can also comprise further sequences. These are preferably plasmid or vector sequences. Polynucleotides in the sense of the invention can be ribonucleotides, deoxyribonucleotides or derivatives thereof. The term encompasses DNA and RNA molecules in single-strand or double-strand form. The DNA can be both cDNA and genomic DNA. The term also includes the known types of modifications of the Polynucleotides, eg methylation, "capping", base substitution with natural or synthetic analogs, intemucleotide modifications with uncharged compounds (eg methyl phosphate, phosphoamidate, carbamate, phosphotriester etc.) or with charged compounds (eg phosphorothioate, phosphorodithioate etc.) or with Bindeglie such as proteins and peptides (e.g. Nucleases, toxins, antibodies, poly-L-lysine, etc.). The term also includes forms with intercalators (e.g. acridine, psoralen etc.), chelators (e.g. with metals, radioactive metals or oxidizing metals etc.), those with alkylating agents and finally with modified bonds (e.g. alpha anomeric nucleic acids etc.).

The term “inner polynucleotide” encompasses any polynucleotide which is intended to serve as a template for RNAi molecules. For the purposes of the invention, inner polynucleotides are preferably DNA molecules or fragments thereof which are transcribed into cells and / or for which there is a corresponding RNA, such as

Genes or cDNAs. Here, cDNAs are particularly preferred, in particular also in

Form of cDNA libraries.

The term “eukaryotic expression control sequence” encompasses each of the cis-regulatory elements which are necessary for the expression of a gene or a cDNA in

Eukaryotes are needed. By definition, eukaryotes include all cells or organisms that - unlike prokaryotes - have a cell nucleus that is well delimited from the cytoplasm by two nuclear membranes. Cis-regulatory elements are DNA sequences with regulatory

Characteristics. These include promoter, enhancer and silencer elements.

Promoter elements mediate the basal expression of a gene, whereas

Enhancer elements increase expression, silencer elements, however, reduce or inhibit expression. The promoter, enhancer and silencer elements interact physically with regulatory proteins

Transcription factors. Transcription factors can influence gene expression in different ways. Some transcription factors, the so-called basal transcription factors, bind to DNA elements such as the TATA box or other so-called "initiator" elements or to neighboring elements

Sequence segments. The basal transcription factors form a complex that ultimately also recruits RNA polymerase, a DNA-dependent RNA-synthesizing enzyme that mediates the actual transcription.

Transcription factors that bind to enhancer elements ensure fast

Formation of a stable complex of the basal transcription factors by, for example, directly or indirectly via further proteins, the so-called co-

Recruit factors and stabilize the complex initiated in this way.

In contrast, transcription factors that bind to silencing elements negatively interfere with the formation of a complex of the basal transcription factors.

However, a number of transcription factors only change the structure of the

DNA and thereby usually brings distant cis-regulatory sequences in close proximity to one another, so that transcription factors binding therein can interact physically with one another. Understandably, such cis-regulatory elements or the transcription factors that bind to them can also

Affect gene expression as enhancer or silencer elements. For the tissue-specific expression of a gene, the presence and the architecture of enhancer and silencer elements in a gene locus, on the other hand the tissue-specific expression of the cis-regulatory elements

Elements binding transcription factors responsible. Under the term

“Expression control sequence” in the sense of the invention is therefore to be understood as a DNA sequence which comprises various of the previously described cis-regulatory elements which are sufficient for the expression of the latter

To mediate expression controUsequence associated polynucleotide and which are normally involved in vivo in mediating the tissue specificity of gene expression. The organization and functioning of eukaryotic

Expression control sequences are, for example, in Genes V, Lewin B., Oxford

University Press (1994). Preferred expression control sequences in

The meaning of the invention are strong promoters, by means of which sufficiently long transcripts of the polynucleotide can be formed in sufficient quantity to enable the bimolecular assembly of the single-stranded RNA molecules into a functional RNAi molecule. The transcription efficiency of

Expression control sequences can be found in known test systems, e.g. through Northern

Analyzes or RNase protection experiments can be checked. Particularly preferred

Expression control sequences within the scope of the invention are described in more detail below.

The term "polyadenylation sequence" refers to a polynucleotide sequence that mediates the processing of the 3 'end of the eukaryotic mRNA as set out below. To process the transcript, a must

Polyadenylation (poly-A) signal present. Polyadenylation refers to that which occurs after the synthesis of almost all eukaryotic mRNAs at the 3 'end

Attachment of approx. 20-250 adenyl residues by a core poly (A) synthetase.

These residues are also referred to as poly (A) tails. A sequence conserved in many genes, AATAAA, is responsible for the processing of the mRNA and is located 6-30 bases in the 5 'direction in front of the polyadenylation site. Other, either U-rich or G + U-rich, less conserved sequences in the 3 'direction behind the polyadenylation site are also required for the correct processing of the 3' end of an mRNA. The importance of polyadenylation should lie in the stabilization of the mRNA. Whether a particular nucleotide sequence can act as a polyadenylation sequence can easily be determined by the person skilled in the art using known techniques. Particularly preferred Polyadenylation sequences within the scope of the invention are SV-40 and BGH

Polyadenylation.

The polynucleotide described above containing an inner polynucleotide is used in the

Sequence also referred to as the bi-promoter expression vector.

By using the eukaryotic polynucleotides, e.g. in the form of eukaryotic expression plasmids, the formation of double-stranded RNA (dsRNA) is made possible directly in the target cell for the first time. Based on the prokaryotic expression system by Kamath (2000), which is a prokaryotic

Expression system for RNAi molecules, the dsRNA is described here by a

Expression unit generated with two oppositely arranged eukaryotic promoters. The two promoters preferably flank a complete or partial cDNA sequence. However, the polynucleotide according to the invention contains

Contrary to the expression system from Kamath, loc. cit. eukaryotic

Expression control sequences, for example CMV promoters or tk-

Promoters. For such promoters there is functionality in one

The arrangement has not yet been described. The promoters must be at least strong enough so that from both sides, i.e. of 5 'and of 3' sufficiently long transcripts can be produced in sufficient quantity, so that the individual can be assembled in a bimolecular reaction

Single stranded RNA molecules (ssRNAs) in the cell to enable a dsRNA.

The promoters should therefore preferably be strong promoters, e.g. CMV

Promoters. A single cDNA or an entire cDNA library can be cloned between the two promoters (see FIG. 1). Both can

Promoters can be regulated. If they are regulated according to the same principle, the dose of the expressed dsRNA can be regulated. Will they be different

Regulated in principle, the polynucleotide according to the invention can also be converted, inter alia, into a multifunctional plasmid which can be used both for the expression of sense and / or antisense RNA and of dsRNA - depending on the selected one

Promoter and principle of regulation. For this, e.g. directional cloning of the cDNA may be required. An example of such an expression plasmid is shown in FIG

1 shown. Such an embodiment of the polynucleotide according to the invention is particularly preferred.

Inter alia, the invention is based on the unexpected finding that the

Polyadenylation sequences in the polynucleotides according to the invention outside the expression control sequences must be localized to ensure adequate expression of the inner polynucleotide.

Publications in which the RNA mechanism for inhibiting the

Gene expression in plants has been studied, however, suggest that the

Polyadenylation sequences between expression control sequence and inner

Polynucleotide should be localized. The reason for this is that dsRNA molecules, which also include expression control sequences, are still unclear

Mechanism with the function of the expression control sequences of target genes could interfere and thus observed effects on the actual RNAi

Effect would be attributed. Another surprising finding, which in the

The connection with the application was made that strong promoters, such as CMV promoters, are preferably suitable for the polynucleotides according to the invention. Based on the state of the art, the person skilled in the art would assume that strong promoters in particular cannot be used in a suitable way in a bipromotor construct, since they would compete for the factors regulating the transcription and thus ultimately no or only a very inefficient transcription would be possible ,

The polynucleotides according to the invention can advantageously be introduced into eukaryotic host cells by the methods known in the prior art, which are also described in more detail below.

Surprisingly, it was found in the findings on which the invention is based that the polynucleotides according to the invention, even after they have been introduced into the cell, are able to form RNAi molecules which specifically interfere with the biological activity of the target gene against which they are directed , The efficiency of the introduction can vary depending on the used

Insertion procedures can be maximized. In addition, is in the state of the

Techniques known in the art enable efficient purification of DNA molecules, as a result of which undesired non-specific effects from contaminating substances no longer occur. It is particularly advantageous that a large number of different polynucleotides according to the invention, which can be used, for example, in screening processes, can be produced by coding

Polynucleotides from existing libraries, e.g. cDNA libraries, by molecular cloning into the polynucleotides according to the invention as internal

Polynucleotides can be included. Another advantage of Polynucleotides according to the invention consist in the fact that these molecules can easily be used in screening processes, in particular in so-called high throughput screening (HTS). DNA polynucleotides are particularly suitable for these high throughput processes since a number of In addition to ensuring a specific RNAi effect and high efficiency in introducing different polynucleotides into the host cells, it is also important in such processes to counter the structure, ie the nucleic acid sequence of the RNAi molecules or the target genes The use of the polynucleotides according to the invention ensures this, since in particular the DNA polynucleotides can easily be sequenced using known and automated methods driving enables the detection of genes, the inactivation of which leads to detectable changes in the target cell. A suitable screening method is described in another context in WO01 / 48239. The use of RNAi in a screening method has the advantage that a reduction / switch-off of the gene expression of a target gene can be detected. Compared to the use of antisense RNA, the likelihood of blocking gene expression is increased, since antisense molecules are often unable to hybridize with their target RNA due to cumbersome secondary structures. In addition, sequences from the 3 ^' region of genes would preferably be cloned when producing a gene bank with antisense RNA. This prevents efficient antisense effects, which can preferably be achieved in the region of the 5 ^' mRNA sequences (in the region of the start codon). In contrast to inducers for RNAi, antisense molecules, which are mostly used as oligonucleotides, must be present in significantly higher amounts (μM concentrations to nM). The use of lower concentrations of RNA leads to an increase in transfection efficiency. The transfection of the polynucleotides according to the invention, for example in the form of plasmids, also facilitates the introduction of the dsRNA into the cell, since the RNA is expressed in the target cell. Previously, the RNA had to be obtained by in vitro transcription reactions, the complementary RNA strands purified or hybridized if necessary and then in various ways (such as by calcium phosphate transfection, lipofection or microinjection) into a target cell or one Target organism are introduced. Last but not least, the polynucleotides according to the invention can also be used to treat and / or prevent

Diseases are used that are based on incorrect regulation of the target genes of the ds RNA (RNAi) molecule. Here, the polynucleotides can be used as DNA molecules, for example in the context of gene therapy approaches. Diseases preferred within the scope of the invention are described in more detail below.

In a preferred embodiment of the polynucleotide according to the invention, the inner polynucleotide comprises at least 50 nucleotides.

In a further preferred embodiment of the invention, the inner polynucleotide comprises a cDNA molecule or a fragment thereof.

The term "fragment of a cDNA molecule" includes cDNA molecules which have the characteristic and specific components of the inner polynucleotide. These fragments are preferably sufficiently long that the RNAi molecules they form can specifically inhibit the function of the target gene. Polynucleotides which comprise at least 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides or 1000 nucleotides are particularly preferred. This fragment preferably contains no start codon.

It follows from the statements made above that in a particularly preferred embodiment of the polynucleotide according to the invention the cDNA molecule or fragment thereof comes from a library of cDNA molecules. The term “library of cDNA molecules” includes gene banks which contain mRNA sequences in the form of cDNA. Preferred here are cDNA libraries from eukaryotic organisms, in particular from mammals and preferably from humans. The preparation, isolation and cloning of cDNA molecules or cDNA fragments fails cDNA libraries are known to the person skilled in the art and are described, for example, in standard molecular biology textbooks, such as Sambrook et al. or in Ausubel et al. In a further preferred embodiment of the invention, the first and the second expression control sequence are identical to one another or different from one another.

In a further preferred embodiment of the invention, the expression control sequence is selected from the group consisting of CMV promoter, thymidine kinase promoter, SV40 promoter or PGK promoter, α-myosin heavy chain promoter.

In a preferred embodiment of the invention, the first and the second expression control sequence are constitutively active.

The term "constitutively active expression control sequence" denotes an expression control sequence which is activated by the transcription machinery already contained in the cell.

In another preferred embodiment of the invention, the first and the second expression control sequence are inducible.

The term "inducible expression control sequence" denotes an expression control sequence which, by adding an inducer, for example certain chemicals (e.g. Cu ⁺⁺ ions, methanol etc.) or by other influences such as heat, the transcription of a gene which is functionally linked to this expression control sequence induced.

If the expression control sequences are regulated according to the same principle, the dose of the expressed dsRNA can be regulated. If they are regulated according to different principles, the bipromotor plasmid of the invention can be converted into a multifunction plasmid which can be used both for the expression of sense and / or antisense RNA and of dsRNA - depending on the chosen promoter and regulation principle. Directed cloning of the cDNA may be required for this. An example of this is the Ecdyson system (Invitrogen, Karlsruhe).

In a particularly preferred embodiment of the invention, the inducible first and second expression control sequence is selected from the group consisting of tetracycline inducible promoters, metallothionine promoters and ecdysone inducible promoters (Gossen and Bujard, 1992; Clontech, Tet-System; Acra et al., 1998; Thummel, 2002).

In a preferred embodiment of the invention, the first and the second polyadenylation sequence are identical to one another.

In another preferred embodiment of the invention, however, the first and the second polyadenylation sequence are different from one another.

The invention also relates to a method for producing a double-stranded polynucleotide comprising the steps:

(a) Linking a single-stranded first DNA molecule which at the 5 'end comprises a recognition sequence for a restriction endonuclease, at its 3 ^' end with a second oligonucleotide whose 5 'end is phosphorylated, the second oligonucleotide (i) comprising a sequence, which allows the linkage to the first DNA molecule and (ii) comprises at the 3 ^' end a sequence of at least 5 nucleotides which allows the formation of a hairpin-shaped secondary structure ("stem loop") structure;

(b) Synthesis of a second DNA molecule, the first single-stranded DNA molecule serving as a template for the synthesis of the second DNA molecule and the second DNA molecule serving as a primer starting from the 3 ^' end of the second oligonucleotide forming a hairpin-shaped secondary structure, at the end the synthesis is a double-stranded DNA molecule consisting of the first DNA molecule, the second oligonucleotide and the second DNA molecule;

(c) denaturing the double-stranded DNA molecule thus obtained; and

(d) Synthesis of a third single-stranded DNA molecule using a third oligonucleotide comprising a sequence identical to the first oligonucleotide, the second single-stranded DNA molecule from step (c) serving as a template and the second and third single-stranded DNA molecules as a double strand at the end of the synthesis. All previously made term definitions meet these and all subsequent ones

Embodiments mutatis mutandis too.

In addition to the steps explicitly mentioned above, the term “production” also includes additional steps, such as pretreatments of the starting material or

Further treatments of the end product. Pretreatment methods are explained in more detail below in the description of the preferred embodiments. Further processing methods include, for example, chemical

Modification or other common formulation and / or assembly steps.

This includes in particular purification steps,

Enrichment steps and the subsequent provision of the polynucleotides produced by the method according to the invention, e.g. in suitable

Containers, packaging etc.

The term "link" encompasses a process in which between two neighboring

Nucleic acid bases a chemical bond is made. It is preferably a 5'-3'-phosphodiester bond in the sugar phosphate backbone of the

Polynucleotide between two neighboring bases. Protocols for the linking of polynucleotides, also referred to as ligation, have been described in the literature (Sambrook et al .; Ausubel et al.).

The term "restriction endonuclease recognition sequence" includes one

Area of a defined base sequence which is specifically recognized by a restriction endonuclease and which may be cleaved. The recognition sequence for one

Restriction endonuclease can range from 4 to 10 base pairs. As part of the

According to the invention, such recognition sequences are preferred which comprise 6 base pairs and more.

The term "second oligonucleotide" encompasses an oligonucleotide whose 5 'end is phosphorylated, the second oligonucleotide also comprising a sequence which allows the linkage to a first DNA molecule and one at the 3 ^' end

Sequence of at least 5 nucleotides comprises, which allows the formation of a hairpin-shaped secondary structure ("stem loop") structure.

In a preferred embodiment, the second oligonucleotide has a free, single-stranded end (10 to 50 nucleotides) at the 5 'end. This overhang serves as a recognition motif for the T4 RNA ligase.

In a further preferred embodiment, the second oligonucleotide has a free single-stranded 3 'end, consisting of an overhang of 3 to 5 guanine Bases, on. A known 3 'region is obtained by adding 3 to 5 cytosine bases to the 3' region of the single-stranded first DNA molecule. A second oligonucleotide is hybridized to these, which has a single-stranded 3 '

Contains a range of 3 to 5 guanine bases. After hybridization of the first single-stranded DNA molecule with the second oligonucleotide, the gap is closed with a T4 DNA ligase.

The advantage of this system lies in the improved efficiency of the substrate conversion by using a T4 DNA ligase instead of a T4 RNA ligase as well as

Attachment of the primer by hybridization.

Phosphorylation of polynucleotide ends has been described in the literature (Sambrook et al .; Ausubel et al.). The length of the second oligonucleotide is between 10 and 150 nucleotides, preferably 20 to 100 nucleotides. Suitable techniques for the design and manufacture of suitable, specific oligonucleotides are known to those skilled in the art. Preferred oligonucleotides which can be used in the process according to the invention are described in more detail below and in the examples. The term "hairpin-shaped secondary structure" or "stem-loop" structure denotes a double-helical region which is formed via intramolecular base pairing between adjacent (inverted) complementary sequences of a single-stranded DNA or RNA. This structure thus enables the oligonucleotide end to be refolded itself. In the oligonucleotides used in the method according to the invention, the hairpin loop forms after hybridization of the 3 'end of the oligonucleotide with its 5' end. In order for the two ends to hybridize with one another, the hybridized portions, ie the 3 'and 5' ends of the oligonucleotide, must be used , include at least enough nucleotides to allow specific hybridization, and the segment between the two ends must include enough nucleotides to spatially form a hairpin loop.

The term "synthesis" encompasses the linking of nucleotides to polynucleotides. In the method according to the invention, the synthesis is preferably mediated by polymerases, the polynucleotides preferably being DNA or cDNA. The synthesis of polynucleotides has been described in the literature (Sambrook et al., Ausubel et al.)

The term "denaturation" means treatment that

Hydrogen bonds between the strands of a double-stranded

Polynucleotide triggers. Denaturation is also known as melting.

Denaturation of polynucleotides can be achieved, for example, by increasing the

Temperature or by low salt concentrations. Denaturation is preferably achieved by increasing the temperature. As is known to the person skilled in the art, the melting temperature of the respective polynucleotides is a decisive parameter which, among other things, is influenced by the relative GC content of the polynucleotides. The melting temperature for polynucleotides in solution is approximately in the range of 85-95 ° C. Methods and formulas for determining the

Melting temperatures are described in the prior art.

The term "third oligonucleotide" includes an oligonucleotide that is one of the first

Oligonucleotide has identical sequence. With the first oligonucleotide in

Within the scope of the invention, an oligonucleotide is referred to which can hybridize specifically with the 3 'end of eukaryotic mRNAs. Preferred here are oligo dT primers which can hybridize with the poly (A) tail of eukaryotic mRNAs. Oligo-dT primers are described in the prior art. The third

Oligonucleotide is used in the process according to the invention for third-strand synthesis and can be produced by processes known in the prior art. Preferred as the fourth oligonucleotide is a 5 ^' phosphorylated (anti

Haamadelprimer) primer used in the unfolded area

Hairpin structure binds to avoid refolding of the second strand product. Preferably, the 3 'end of the oligo dT region of the first

Bases A, C, G to ensure that the oligo-dT primer is exactly at the transition between the polyadenylation sequence (pA) sequence and 3 'UTR

(untranslated area) of the transcript.

In a step preceding the method according to the invention, which was described above, an oligo-dT primer which is provided with at least one rare restriction site can be used in a cDNA first strand synthesis. The further steps are then preferably carried out as described below: Before the second strand synthesis takes place, a T4-RNA Ligase or a T4 DNA ligase (see above) a special DNA

Hairpin primer ligated to the 3 end of the first strand cDNA. This oligonucleotide

(Primer) is characterized by the following properties: It has a phosphorylated 5 ^' end, a single-stranded region (for example 10-20 bp), which is ligated by T4-RNA ligase (Tessier et al., 1986; Delort et al ., 1989; Edwards et al., 1991; Troutt et al., 1992; Chenchik et al., 1996) and one

Hairpin loop ("stem-loop" structure), which enables the primer end to be folded back onto itself. The “loop” must be greater than 5 bp in order to ensure subsequent amplification of the cloned reaction product in E. coli bacteria. A length of the strain of approximately 6-10 bp is preferred. Examples of

Primers that enable such self-priming are for cellular and viral

Nucleic acids described in the prior art (for beta-globin mRNA (Rougeon and Mach, 1976; Volloch et al., 1994); for BC1 mRNA (Shen et al., 1997); for viral

Nucleic acids (Salzman and Fabisch, 1979; Baroudy et al., 1982; Lin and Levin,

1998). After the ligation one becomes at the 3Εnd of the refolded primer

Second strand synthesis performed. The result is an uninterrupted, unilaterally covalently closed paired DNA second strand synthesis product. To

Denaturation is carried out using an identical to the original oligo dT primer

Primers the third strand synthesis. To avoid refolding of the second strand product, a further 5 ^' phosphorylated (anti-hairpin primer) is preferred.

Primer used that the above corresponds to the fourth oligonucleotide. In this preferred embodiment, however, the third-strand synthesis product should still be treated with T4 DNA ligase before it is cloned into a common expression plasmid. Such a construct is also called as

Hairpin expression vector (see for example Figure 2).

Corresponding common expression plasmids are known in the prior art. The

Cloning into the expression plasmid takes place via the rare restriction site of the oligo-dT primer. This gene bank is then preferred in recombination defects

E. coli bacteria such as B. "E. coli acid "transfected.

The methods according to the invention advantageously provide polynucleotides which allow the production of single-stranded RNA molecules in equimolar amounts. For polynucleotides that have two expression control sequences for strand and counter strand of the RNAi molecule, for example, flanking Sequences the transcription undesirably in different

Influence dimensions. When transcribing the by the invention

However, such undesirable influences can be avoided using process-produced polynucleotides. A linear single strand of RNA is first of all generated from the polynucleotides produced by the methods according to the invention

Molecule formed, which comprises the two strands of the RNAi molecule. The two

Strands of the RNAi molecule initially hybridize intramolecularly, but remain connected via the hairpin loop. The advantages of the polynucleotides produced by the method according to the invention when used in the

Analysis of therapeutically or diagnostically relevant target genes, in screening

Procedures, in high-throughput processes and in the treatment and prevention of

Diseases mutatis mutandis correspond to the advantages listed in connection with the polynucleotides according to the invention.

In a preferred embodiment of the invention, the single-stranded first DNA molecule of the method according to the invention is produced by:

(a) Synthesis of a single-stranded first DNA molecule using a first oligonucleotide comprising an oligo-dT sequence and at the 5 'end of the oligo-dT sequence a recognition sequence for a restriction endonuclease, an RNA polyadenylated at its 3' end Molecule of a single species serves as a template and the first oligonucleotide is able to hybridize to the 3 'end of the polyadenylated RNA molecule;

(b) removing the polyadenylated RNA molecule; and

(c) Provision of the first single-stranded DNA molecule.

The term "first oligonucleotide" includes an oligonucleotide that can (specifically) hybridize to the 3 'end of eukaryotic mRNAs. The first oligonucleotide is preferably an oligo-dT primer which can hybridize with the 3 'end of the polyadenylated mRNA, the poly (A) tail of the mRNA, and which at the 5' end has at least one rare restriction site, for example an interface from 6 or more nucleotides. The design and production of specifically hybridizing oligonucleotides is known to the person skilled in the art and is described in the prior art. Melting temperatures of oligonucleotides can be calculated using known computer programs. The first oligonucleotide preferably contains at the 3 'end of the oligo-dT-

The bases G, A, C.

The term "polyadenylated RNA molecule of a single species" means one or more identical mRNA molecules. This includes mRNA

Molecules from non-vertebrates or vertebrates. MRNAs are preferred

Mammals, especially human mRNAs. mRNAs can be made up of cells,

Body fluids (such as lymph, serum, plasma, urine, spinal fluid etc.),

Tissue biopsies etc.

The term "hybridization" means within the scope of this invention

Hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrook (Molecular

Cloning, A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press,

Cold Spring Harbor, NY) or the examples described.

The term "remove" includes the separation and removal of the building blocks of the polyadenylated RNA molecule. For example, in mRNA / cDNA hybrids, the mRNA molecules can be removed by incubation with RNases or by alkaline hydrolysis. Incubation with RNase H is preferred.

The term "supply" includes purification methods and

Enrichment methods of (single-stranded) DNA molecules. These are described, for example, in Sambrook et al. and in Ausubel et al. described.

In a preferred embodiment of the invention, the polyadenylated RNA molecule is obtained by extracting mRNA from cells, tissues or complete organisms or by transcription of cDNA molecules which are contained in libraries of cDNA molecules.

Methods for the extraction of mRNA from cells, tissues or organisms and the transcription of cDNA molecules are described in the prior art (Sambrook et al., Ausubel et al.)

The invention further relates to a method for producing a mixture of double-stranded polynucleotides comprising the steps:

(a) Linking of single-stranded first DNA molecules which at the 5 ^' end have a recognition sequence for a restriction endonuclease at the 3 ^' ends of each with a second oligonucleotide, the 5' end of which is phosphorylated, the second oligonucleotide comprising (i) a sequence which allows the linkage to the first DNA molecule and (ii) one at the 3 ^' end Sequence of at least 5 nucleotides comprising which allows the formation of a hairpin-shaped secondary structure ("stem loop") structure;

(b) Synthesis of second DNA molecules, the first single-stranded DNA molecule serving as a template for the synthesis of the second DNA molecule and the second DNA molecule starting from the 3 ^' end of the second oligonucleotide forming a hairpin-shaped secondary structure, serving as a primer, on At the end of the synthesis, a double-stranded DNA molecule consisting of the first DNA molecule, the second oligonucleotide and the second DNA molecule is present;

(c) denaturing the double-stranded DNA molecule thus obtained; and

(d) Synthesis of third single-stranded DNA molecules using a third oligonucleotide each, which comprises a sequence identical to the first oligonucleotide, the second single-stranded DNA molecule from step (c) serving as a template and the second and third single-stranded DNA molecules present as a double strand at the end of the synthesis.

All previously made definitions of terms apply to this and all subsequent embodiments mutatis mutandis.

The term "mixture of double-stranded polynucleotides" denotes a multiplicity of double-stranded polynucleotides according to the invention, which comprise identical or different nucleic acid molecules.

In a preferred embodiment of the invention, the single-stranded first DNA molecules of the method according to the invention are produced by:

(a) Synthesis of single-stranded first DNA molecules using first oligonucleotides comprising an oligo-dT sequence and at the 5 'end of the oligo-dT sequence a recognition sequence for a restriction endonuclease, with polyadenylated RNA molecules at their 3' end different species serve as a template and the first oligonucleotides in the Are able to hybridize to the 3 'ends of the polyadenylated RNA molecules;

(b) removing the polyadenylated RNA molecules; and

(c) Provision of the first single-stranded DNA molecules.

The term “polyadenylated RNA molecules of different species” in the context of the invention denotes structurally different mRNA molecules. Mixtures of mRNAs which can be obtained from gene banks, cells or cell lines are preferred. The mRNAs can preferably be obtained from non-vertebrates or vertebrates, in particular from mammalian cells. Human mRNAs are most preferred.

In a preferred embodiment of the method according to the invention, the polyadenylated RNA molecules are obtained by extraction of mRNA from cells, tissues or complete organisms or by transcription of cDNA molecules which are contained in libraries of cDNA molecules.

In a preferred embodiment of the methods of the invention, the restriction endonuclease recognizes a sequence of at least 6 nucleotides.

In a particularly preferred embodiment of the methods according to the invention, the rarely cleaving restriction endonucleases are selected from the group consisting of: Xho I, Not I, Xba I, Bgl II, Asp 718, Sal I, Sac I, Sfi I.

In a further preferred embodiment of the method according to the invention, the sequence from (a) (i) which permits the linkage is a 5 'single-stranded region (overhang) which serves as the recognition region for the T4 RNA ligase.

In a still further preferred embodiment of the method according to the invention, the sequence from step (a) (i) which allows the linkage is a single-stranded 3 'region from 3 to 5 guanine bases which, after hybridization with the 3' region of the single-stranded first DNA Molecule is closed by a T4 DNA ligase. In a preferred embodiment of the methods of the invention, the

Sequence from step (a) (ii) showing the formation of a hairpin-shaped secondary structure

("Stem loop") allows at least 5, 6, 7, 8, 9, 10 or up to 100 nucleotides in length.

In a preferred embodiment of the method according to the invention, a fourth oligonucleotide is added in step (d), which is phosphorylated on 5 ^' and comprises a sequence complementary to the second oligonucleotide. For the purposes of the invention, the term “fourth oligonucleotide” encompasses an oligonucleotide which is complementary to the second oligonucleotide described above. As already mentioned, this second oligonucleotide has a sequence which allows the formation of a hairpin-shaped secondary structure. The fourth oligonucleotide can be used in the context of the invention to avoid refolding of the second strand product. The fourth oligonucleotide is preferably an oligonucleotide complementary to the hairpin primer and phosphorylated at the 5 'end. The preparation and the phosphorylation of such an oligonucleotide are known to the person skilled in the art.

The invention further relates to a method for producing a vector or a mixture of vectors, the method comprising the additional step of cloning the heterologous polynucleotides produced into a suitable vector.

The term "vector" refers to prokaryotic or eukaryotic cloning and / or expression vectors. Examples of prokaryotic vectors are chromosomal vectors such as bacteriophages (eg bacteriophage lambda, P1) and extrachromosomal vectors such as plasmids, circular plasmid vectors being particularly preferred. Suitable prokaryotic vectors are described, for example, in Sambrook et al., Chapters 1 to 4. The vector according to the invention can also be a eukaryotic vector, for example a yeast vector or a vector suitable for higher cells, for example a plasmid vector viral vector, a plant vector, etc. Examples of such vectors are also in Sambrook et al. (Chapter 16).

The term "mixture of vectors" includes several identical or different ones

Vectors. The vectors can be the same or different according to the invention

Include polynucleotides. Process for producing a vector or

Mixtures of vectors and the methods of cloning polynucleotides into such vectors are described in Sambrook et al. and Ausubel et al. described. The

Construction of the vector according to the invention advantageously allows the polynucleotides according to the invention to be cloned and / or expressed in eukaryotic cells.

The term "heterologous polynucleotides" means in the context of

Invention that the specific and characteristic components of the

Polynucleotides of the invention (such as the polyadenylation sequences or the promoters) can be from different species.

In a preferred embodiment of the method according to the invention, the polynucleotide or the vector is then treated with a T4 DNA ligase.

The invention further relates to a vector containing a polynucleotide according to the invention or a polynucleotide which is produced by a method according to the invention.

In a further embodiment, the invention relates to a host cell which contains a vector according to the invention.

According to the invention, the term “host cell” includes both prokaryotic and eukaryotic host cells. Prokaryotic host cells include, for example, E. coli, Streptomyces, Bacillus or Salmonella cells. E. coli "SURE" cells are particularly preferred here. Eukaryotic host cells include fungal cells, for example yeast cells, plant cells, insect cells such as Drosophila or SF9 cells, animal cells, in particular mammalian cells. 293 cells, NIH3T3 cells, BHK are preferred here Cells, CHO K1 cells, and HeLa cells. The cultivation of these cells is

Standard in cell biology and e.g. described in Sambrook et al. or Ausubel et al ..

The invention also relates to a method for producing a double-stranded RNA which comprises the step of bringing a polynucleotide according to the invention or a polynucleotide produced by a method according to the invention into contact with a protein or protein mixture under conditions which allow the synthesis of a double-stranded RNA , includes. All previously made definitions of terms apply to this and all subsequent embodiments mutatis mutandis.

The term "contacting" encompasses all types of physical or chemical interactions between the polynucleotides and the protein or protein mixture. For contacting, the polynucleotide can be in solution in a suitable liquid, for example in a buffer, the liquid also containing the protein or protein mixture. The protein or protein mixture can be introduced into the liquid before or after the polynucleotide. A suitable liquid in the sense of the invention also contains the necessary components which are required for the synthesis of the RNA. These are preferably the ribonucleotides and buffer substances, ions etc. which the protein or protein mixture requires in order to catalyze the synthesis of the RNA. Suitable liquids are known and described in the prior art. Instead of a liquid, gels and gel-like liquids can also be used. In the context of the invention, the term “protein or protein mixture” means a protein or protein mixture which is able to catalyze the synthesis of RNA molecules. Such proteins are preferably RNA polymerases. Suitable RNA polymerases are described in more detail below. In addition to the RNA polymerases, a mixture of proteins which is used in the method according to the invention can also contain proteins which regulate the polymerases or which additionally chemically modify the RNA transcripts, such as enzymes which are involved in the polyadenylation described above. The inventive method described here is preferably carried out in vitro, ie in a cell-free system. In a preferred embodiment of the method according to the invention, the protein or protein mixture contains T7 polymerase, T3 polymerase or SP6 polymerase.

Polymerase. Properties and applications of T7 polymerase, T3 polymerase or SP6 polymerase are described in the prior art.

The invention further relates to a method for producing a double-stranded RNA, the method comprising the steps:

(a) introducing a vector of the invention or a vector obtainable by the method of the invention into a host cell; and

(b) Culturing the host cell for a period of time and under conditions that allow the synthesis of double-stranded RNA from the vector in the host cell.

The term "introduction" encompasses all types of physical or chemical interactions between the polynucleotides and the cell or the cellular components. For introduction, the polynucleotide can be in solution in a suitable liquid, for example a nutrient medium for the cell, this nutrient medium then being brought into contact with the cell, for example by incubating the cell in this medium. Instead of a liquid, gels and gel-like liquids can also be used. In addition to the introduction of the polynucleotides into the cell, the term introduction also optionally includes the integration of the polynucleotide into the genome of the host cell. Examples of methods which are suitable for introducing nucleic acids are precipitation transfection, such as, for example, calcium phosphate or RbCI precipitation transfection, transfection by means of liposomes, transfection by means of macromolecular polymers, for example fullerenes, electroporation methods or transfection by retrovection or recombinant techniques for integration into the cellular genome. Depending on the method, it is possible that the nucleic acids have to be linked to other nucleic acid molecules. Examples of these are plasmids which contain the nucleic acid molecules or retroviral genomes in which the nucleic acids have been integrated. Nucleic acid molecules can also be integrated into the cellular genome after introduction into the cell. According to the invention, the term “cultivating the host cell” is understood to mean all measures which are necessary to ensure the vitality

Growth of host cells and the ability to synthesize RNAs in the

To maintain host cells. This is preferably ensured by a culture medium which contains nutrients and, if appropriate, growth and

Contains survival factors. Preferred culture conditions are in the prior art

Technology or described in more detail in the examples (e.g. Current Protocols).

Vectors containing the polynucleotide of the invention can be obtained via conventional transfection methods such as e.g. by calcium phosphate transfection

(Ui-Tei et al., 2000); Lipofection (Lin et al., 2001), microinjection (Tabara et al., 1998),

Electroporation, viral transfer or other transfection methods into which cells are introduced. The cultivation of these cells under conditions that a

Allow expression of the polynucleotides / vectors according to the invention, and

Methods for the detection of the polynucleotides produced are standard methods in cell biology or molecular biology and are extensively described in Sambrook et al. or

Ausubel et al. The statements made about the advantages of the polynucleotides according to the invention apply mutatis mutandis to the method described above

The invention additionally relates to a method for the identification and / or production of genes, the inactivation of which leads to detectable changes in the target cell, wherein in addition to the above-mentioned method the following step is included: (c) comparison of the phenotype of the host cell from (b) with a host cell into which no vector or control vector was introduced in step (a). All previously made definitions of terms apply to this and all subsequent embodiments mutatis mutandis.

The term "identification" encompasses the identification of a gene and / or its function (s), which is made possible due to the detectable changes in the target cell resulting from the inactivation of the gene. Such changes can be triggered by the use of RNAi in a screening process in the sense of the invention.

The term "detectable changes in the target cell" refers to changes at the molecular level, as well as changes that change the phenotype the cell, for example the cell morphology. suitable

Analysis methods are known and described to the person skilled in the art. The

Cell morphology can e.g. be examined by means of morphometric methods.

When analyzing gene expression or protein expression come preferred

Methods like Northern analysis, RNase protection experiments, PCR based

Techniques, or Western analyzes for use. These analysis methods can also be integrated into an automated process.

The term “inactivation” in the context of the invention also includes a significantly reduced expression of a target gene which is a detectable change in the

Target cell causes. Comparative tests between treated and untreated target cells can be used to determine whether the expression of a target gene has changed significantly. This is also described in more detail below and in the examples. The observed expression levels can be checked for significant differences using suitable statistical tests. Such statistical

Tests include, for example, the Student's T test, the Chi ² test, and known variations based thereon. The reduction is preferably

Gene expression by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or the cheapest

Fall by 100%. Target gene can be, for example, a cell gene, an endogenous gene, but also a transgene or a gene of a pathogen that is infected with an infection in the

Cell has arrived.

The term "phenotype" denotes the appearance of a cell that is characterized by the

Sum of all characteristics that are anchored in the genes of the cell. The

Phenotype includes all external and internal structures and functions of the cell.

The term “control vector” denotes the vector used for the above-mentioned method, which, in contrast to this, is not an inventive one

Contains polynucleotide. The specificity of the method or of the invention

Polynucleotides or vectors can be checked or ensured by suitable controls in which target cells are transfected, for example with this control vector. The construction and implementation of such control experiments are known to the person skilled in the art. If the applied RNA interference in the target cell leads to a specific, detectable effect that does not occur in a suitable control experiment, this effect can allow conclusions to be drawn about the function of the gene.

The term “target cells” in the context of the invention includes eukaryotic

Cells, especially mammalian cells and preferably human cells, in which specifically suppress or at least reduce the expression of a target gene.

The statements made about the advantages of the polynucleotides according to the invention apply mutatis mutandis to the method described above.

In a preferred embodiment of the method according to the invention, the host cell is a prokaryotic host cell.

In a particularly preferred embodiment of the method according to the invention, the prokaryotic host cell is an E. coli “SURE” cell.

In a preferred embodiment of the method according to the invention, the host cell is a eukaryotic host cell.

In a preferred embodiment of the method according to the invention, the eukaryotic host cell is selected from the group consisting of 293 cells, NIH3T3 cells, BHK cells, CHO K1 cells, and HeLa cells.

In a preferred embodiment of the method according to the invention, at least one protein from the group of proteins which can be activated by double-stranded RNA is inactivated or not present in the host cell.

Studies in mammalian cells have shown that accumulation of dsRNA in mammalian cells often leads to an unspecific response, which results in a general blockage of translation and subsequent apoptosis. Non-specific RNAi effects are attributed, among other things, to the presence of an antiviral mechanism which is widespread in mammalian cells and is also known as the interferon response. Longer dsRNA molecules are inducers of the unspecific dsRNA response, provided that they are at least 30 base pairs long. Cellular proteins secrete the dsRNA and initiate a general inhibition of cellular translation (Terenzi et al., 1999; Williams, 1999). This leads to an unspecific reduction in gene expression. The dsRNA activates two enzymes: PKR, which in its active form phosphorylates the translation initiation factor elF2a, which leads to a shutdown of protein synthesis, and 2 ', 5'-oligoadenylate syntetase, which forms a molecule that activates RNaseL, which degrades mRNAs non-specifically (Elbashir et al, 2001). PKR thus plays as a dsRNA

Sensor and in the initiation of the antiviral response (PKR) (Williams, 1999) (Williams, 1999; Zamanian-Daryoush et al., 1999; Der and Lau, 1995); PKR and apoptosis (Der et al., 1997) (Gil and Esteban, 2000a; Gil and Esteban, 2000b; Gil et al., 2001); PKR and involvement of RNase L (Terenzi et al., 1999; lordanov et al., 2000). The non-specific dsRNA response thus competes with the specific dsRNA response and thereby conceals (overlaps) the desired, specific effect by means of RNA interference (Elbashir et al., 2001). As already explained, in the sequence-specific dsRNA response, the RNA interference (RNAi), the initiating double-stranded RNAs are first broken down into short interfering RNAs (siRNAs). The siRNAs (presumably) provide the sequence information which allows a specific degradation of a specific mRNA. A reduction in the unspecific dsRNA response in the target cell (and at the same time an increased specific dsRNA response by RNAi) can be achieved by:

(a) the generation of a PKR-deficient background in the cell, preferably further dsRNA sensor molecules being switched off, or

(b) blocking the intracellular accumulation of large amounts of long chain dsRNA. This can be done by increased processing to 21-23meres, which are the actual RNAi inducers.

Such processing is made possible, for example, by the co-expression of an RNAi-associated nuclease (Ketting et al., 1999; Filippov et al., 2000; Hammond et al., 2000; Bernstein et al., 2001; Dalmay et al., 2001). The human helicase MOl (Matsuda et al., 2000) is due to sequence homologies as the homolog of the RNAi-associated nucleases Mut-7 (C. elegans; (Ketting et al., 1999)) or Dicer (Drosophila; (Bernstein et al., 2001) For Dicer, the processing of long dsRNA to 21-23meres has been demonstrated biochemically (Bernstein et al., 2001) .The helicase MOl has both a comparable RNAse III activity and a helicase activity, such as it is postulated for an RNAi-enzyme complex (Provost et al., 1999; Bass, 2000) Suitable cell lines in which inhibition of the interferon response has been described are, for example, cell lines from PKR-deficient KO mice (Rivas et al. , 1999), PKR deficiency (lordanov et al., 2001; Khabar et al., 2000) or interferon-resistant cell lines (K562, BJAB;: (Yamamoto et al., 2000)). Deficiency of the interferon response can also be achieved by:

(i) Co-expression of the following inhibitory proteins: E1A fragments (Ackrill et al., 1991) (Quinlan, 1993), HepB virus protein (Foster et al., 1991), tetratricopeptide repeat protein and cochaperone p58 (IPK) (Tang et al., 1999), E3L (Shors and Jacobs, 1997) (Shors et al., 1997) (Rivas et al., 1998) (Shors et al., 1998) or TAR (Park et al ., 1994), or by

(ii) the co-expression following inhibitory RNA ^'s: PKR and small- ^RNA' s (. Clemens et al, 1994), VAI RNA (Svensson and Akusjarvi, 1984; O'Malley et al, 1986; Evstafieva et. al., 1988) (Ghadge et al., 1991; Ghadge et al., 1994; Rahman et al., 1995; Desai et al., 1995; Lei et al., 1998), EBER-RNA ^'s (Clarke et al., 1990; Sharp et al., 1993).

In a preferred embodiment of the method according to the invention, the group of proteins which can be activated by double-stranded RNA comprises protein kinase R (PKR) and RNAse L (loc. Cit).

In a preferred embodiment of the method according to the invention, the activity of the RNAi-enzyme complex is increased.

In the context of the invention, the term “increased” is understood to mean a significantly increased activity of the RNAi complex, which can be demonstrated by methods which are described in the prior art. Whether the observed differences are significant can be determined by known statistical tests determined elsewhere in the description.

In a preferred embodiment of the method according to the invention, the RNAi-enzyme complex has at least one protein which has the biological activity of a protein selected from the group consisting of helicase MOl, nuclease Mut-7 or Dicer (loc. Cit.).

In a preferred embodiment of the method according to the invention, the host cell comprises proteins that inhibit the interferon response.

The term “interferon response” encompasses an antiviral mechanism which is widespread in mammalian cells and which can be attributed to non-specific RNAi effects. In a preferred embodiment of the method according to the invention, the proteins inhibiting the interferon response are selected from the group consisting of E1A, HepB virus protein, tetratricopeptide repeat protein, cochaperone p58 (IPK), E3L, or TAR (loc. Cit.) ,

The invention also relates to a transgenic animal containing a polynucleotide of the invention or a polynucleotide which can be obtained by a method of the invention.

In the context of the invention, the term “transgenic animal” is understood to mean non-human transgenic animals which (i) constitutively or inducibly overexpress the polynucleotides or vectors according to the invention, or (ii) have a conditional and tissue-specific overexpression of the polynucleotides or vectors according to the invention A polynucleotide according to the invention or a vector which contains this polynucleotide can be introduced into animals into a germ line cell, an embryonic cell, stem cell or an egg cell or a cell derived therefrom are analyzed using, for example, known techniques such as Southern blotting in conjunction with suitable samples, and transgenic animals in the context of the invention here include mice, rats, hamsters, dogs, monkeys, rabbits, pigs, C. elegans and zebra fish, preferred are transgenic mice. Mice have numerous advantages over other animals. They are easy to hold and their physiology is considered a model system for that of humans. The production of such genetically manipulated animals is well known to the person skilled in the art and is carried out by customary methods (Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994), Manipulating the Mouse Embryo; A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Joyner, AL (Editor), Gene Targeting, A Practical Approach (1993), Oxford University Press. Constructs can optionally be used to produce the transgenic animals tissue-specific, regulated during development Promoters, cell-specific promoters and / or inducible promoters are used which regulate the expression of the polynucleotide of the invention.

A suitable inducible system is, for. B. regulated the tetracycline

Gene expression such as B. by Gossen and Bujard (PNAS 1992, pages

5547-5551). The transgenic animals according to the invention can be used as a model for

Serve diseases in humans or also in farm animals. The animals can also be useful for diagnosis or early detection of a disease.

The invention also relates to a medicament comprising a polynucleotide of the invention or a polynucleotide obtainable by a method of the invention. All previously made definitions of terms apply to this and all subsequent embodiments mutatis mutandis.

According to the invention, the term “pharmaceuticals” defines substances and preparations made of substances which are intended to heal, alleviate, prevent or recognize diseases, ailments, bodily harm or pathological complaints by application to or in the human body. The polynucleotides of According to the invention, medical and / or pharmaceutical-technical auxiliaries can be added. According to the invention, medical auxiliaries are those substances which are used for the production (as active ingredients) of medicaments in a method according to the invention. provided that they are only required during the process, are subsequently removed or may be part of the medicament as pharmaceutically acceptable carriers, examples of pharmaceutically acceptable carriers are listed below g is optionally in combination with a pharmaceutically acceptable carrier and / or diluent. Examples of suitable pharmaceutically acceptable carriers are known to those skilled in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of detergents, sterile solutions, etc. Drugs comprising such carriers can be made using known conventional methods be formulated. These drugs can be administered to an individual in a suitable dose, for example in a range from 1 μg to 100 mg per day and patient. The administration can be done in various ways, for example directly on the skin, intravenously, intraperitoneally, subcutaneously, intramuscularly, locally or intradermally. Nucleic acids can also be administered in the form of gene therapy. The kind of

Dosage is determined by the attending physician according to the clinical factors. It is known to the person skilled in the art that the type of dosage depends on various factors, e.g. the size, the body surface, the age, the sex or the general health of the patient, but also of the special agent that is administered, the duration and type of

Administration and other medications that may be administered in parallel.

The invention also relates to the use of a polynucleotide of the invention or a polynucleotide obtainable by a method of the invention for the manufacture of a medicament which can be used for the treatment or prevention of diseases.

The term “treatment” here denotes therapeutic measures for combating, inhibiting, eliminating or alleviating diseases, while the term “prevention” denotes measures which serve to prevent a disease so that it does not arise at all.

The term "manufacture" of pharmaceuticals also includes additional steps such as common formulation and / or packaging steps. This includes in particular purification steps, enrichment steps, sterilization processes and the subsequent provision of the polynucleotides produced by the process according to the invention, for example in suitable containers etc. The term also includes the formulation of the polynucleotides produced in suitable dosage forms. These can be injection solutions, liposomes, organic carriers or transport molecules, such as fullerenes, capsules, tablets, and other known suitable administration forms for polynucleotides. The guidelines of the GMP (“Good Manufacturing Practice”) are preferably observed in the production of pharmaceuticals. The polynucleotides of the invention can preferably be used for gene therapy in that they are introduced into the cells of a target organism be introduced. The polynucleotides of the invention can be viral

Vectors are cloned which mediate the transfer of the sequences coding for the double-stranded RNA into replicating host cells. Suitable viral

Vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus,

Vaccinia virus, poliovirus and the like. Alternatively, the polynucleotides of the

Invention can be transferred into cells by non-viral gene therapy techniques, including receptor-mediated, targeted DNA transfer using

Ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection,

Membrane fusion or direct microinjection. These methods and variations thereof are suitable for both ex vivo and in vivo gene therapy. Protocols for

Gene therapy is described in Gentherapy protocols, Robbins, Paul D. (Editor),

Human Press, Totawa NJ. (1996).

In a preferred embodiment of the use of the polynucleotide according to the invention, the disease is selected from the group: cancer, diseases of the cardiovascular system, diseases of the skin, diseases of the internal organs, metabolic disorders, neurological diseases or disorders or disorders of the immune system, degenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, reperfusion damage, stroke and alcohol damage to the liver, tumor diseases such as leukemia, carcinoma or sarcoma, autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, diabetes lupus, viral diseases such as hepatitis or influenza. The symptoms of such diseases are described in detail in clinical lexica, such as Pschyrembel or Stedman, and can easily be recognized by a person skilled in the art.

In a further preferred embodiment of the method according to the invention described above, this further comprises the formulation of the polynucleotide obtained by the method according to the invention with a pharmaceutically acceptable carrier, excipient and / or diluent. The figures show:

1: schematic exemplary representation of a bi-promoter

Expression Vector:

PCMVI and PCMVI I show the position of the opposing promoters for the transcription of the opposing transcripts of the target gene (X); the resulting sense and antisense transcripts are shown below; bla: ampicillin resistance; pA: poly-A sequence; ori: origin of replication

2: schematic exemplary representation of a hairpin

Expression Vector:

PCMV shows the position of the promoter for the transcription of the opposite transcripts of the target gene (X); below are the resulting sense and antisense transcripts linked by the hairpin sequence; bla: ampicillin resistance; pA: poly A sequence; ori: origin of replication

3: schematic example of a course diagram for the construction of a hairpin expression vector:

The flow chart shows the individual steps in the construction of a hairpin expression vector

After attachment of the RNAi oligodT primer with restriction site (first oligonucleotide), the first strand (first single-stranded DNA molecule) is synthesized (1). The polyadenylated RNA molecule is removed (by RNAse or alkaline lysis) (2). The second strand (second DNA molecule) can be synthesized by ligation of a hairpin primer (second oligonucleotide a) by T4-RNA ligase and subsequent synthesis by DNA polymerase (3a). Alternatively, the first strand can be extended by an oligo-dC sequence with terminal transferase or a suitable reverse transcriptase before the alternative hairpin primer (2nd oligonucleotide b) is ligated to the first strand by T4-DNA ligase (3b). The double-stranded DNA molecule is then denatured (4) and the third strand (third single-stranded DNA molecule) Attachment of the 3.0 ligonucleotide by a DNA

Polymerase (preferably thermostable) synthesized (5). If necessary, an internal anti-hairpin primer is used to avoid intramolecular refolding. The strand gap can pass through

T4 DNA ligase are closed. After restriction cleavage (in Fig.

3, for example EcoRI), the construct obtained in this way can be inserted into a suitable expression vector regardless of the orientation (6). When expressed, the antisense sequence appears in the

Transcript always first.

Fig. 4: schematic representation of the vector ptwopA

5: Extinction of GFP expression after insertion of an inverted SV40-poly-A fragment between promoter and GFP reading frame: fluorescence microscopic images of 293 cells 24 hours after the transfection. Each includes phase contrast microscopic documentation of the same field of view (20X magnification). A: Transfection of pEGFP-N2, B: Transfection of ptwopA

6: schematic representation of the vector pΔBi-CMV-GFP:

PCMVI and PCMVI I shows the position of the opposite promoters for the transcription of the opposite transcripts from GFP; bla: ampicillin resistance; pA: poly-A sequence; ori: origin of replication

Fig. 7: Singular transfections of GFP-Bi promoter constructs and a conventional GFP expression plasmid in 293 cells: fluorescence microscopic images of 293 cells 24 hours after the transfection. Below this, the phase contrast microscopic documentation of the same field of view (20X magnification) is shown.

Transfection of pΔBI-CMV-GFP (A), pΔBI-CMV-GFP-INV (B), pBI-GFP (C) and pEGFP-N2 (D).

The figure shows that the transfection of the bi-promoter constructs with the complete GFP reading frame (A and B) is strong reduced GFP expression in 293 cells compared to the conventional expression plasmid pEGFP-N2 (D). The

Transfection of the bi-promoter plasmid pBI-GFP with 5 ^' truncated

Reading frame (C), however, did not lead to GFP-positive cells.

On the other hand, these GFP-positive cells showed after transfection of pΔBI-CMV-GFP (A) and pΔBI-CMV-GFP-INV (B), but also that full-length transcripts were generated by both promoters.

Fig. 8: schematic representation of the hairpin expression vector php-1

9: Transfection of a GFP hairpin construct in 293 cells:

Fluorescence micrographs of 293 cells 24 hours after transfection. Each includes phase contrast microscopic documentation of the same field of view (20X magnification). Transfection of pEGFP-N2 (A), php-1 (B)

Fig. 10: Specific reduction of the firefly luciferase activity in cell extracts from transfected CGR8 mouse embryonic stem cells by pBI-Luc and pLuc-hp

The data refer to three independent transfections. Firefly luciferase activity in the cell extract normalized to Renilla luciferase and expressed as a percentage of the measured activity of the control (pcDNA3.1Δneo). The constructs pBI-Luc and pLuc-hp specifically reduced Firefly luciferase activity to about 60% and 50% of the control, respectively. The GFB-directed dsRNA vectors pBI-GFP and php-1 did not reduce the Firefly luciferase activity compared to the control. The examples illustrate the invention.

Unless otherwise stated, the tests were carried out according to "Current Protocols" (Ausubel et al., 2002).

Example 1: Location of the pA (polyadenylation) signal in the bipromotor construct: GFP expression is switched off when an SV40-polyA fragment is positioned between two promoters.

The aim of this experiment was to insert a polyadenylation signal (SV40 polyA fragment) from an origin vector into a target vector using a conventional eukaryotic expression unit. The polyA fragment was inserted between the promoter and the reading frame of the gene to be expressed (GFP; "green fluorescent protein") in the antisense orientation of the promoter into the expression unit of the target vector. The influence of this fragment on the expression of GFP should be observed target vector.

A BamHI / HindIII fragment (458 base pairs) from the vector pTet-OFF (GenBank accession number: U89929) was inserted into a GFP expression vector pEGFP-N2 (GenBank accession number: U57608) by restriction cleavage with HindIII and BamHI , which contained its SV40 polyA fragment.

The orientation of this fragment relative to the PhCMV promoter in pEGFP-N2 was opposite to the orientation of the fragment in the vector pTet-OFF to the associated promoter. The inserted SV40-polyA ~ fragment (SV40-pA ^' ) was in the indicated orientation between the promoter (PhCMV) and the GFP reading frame. The resulting vector was named ptwopA (Fig. 4). pEGFP-N2 and ptwopA were transfected into 293 cells using conventional calcium phosphate transfection and the expression of GFP was monitored by fluorescence microscopy 24 hours after the transfection (FIG. 5).

The example shows that the transfection of ptwopA did not lead to GFP-positive transfectants, in contrast to that of pEGFP-N2, which led to numerous GFP-positive transfectants. The insertion of the polyA fragment leads to the GFP expression being switched off. This is probably due to processing and polyadenylation of the transcript before the GFP reading frame, mediated by polyA signals of the inserted SV40-polyA fragment, which are also present in the normally non-transcribed strand - for example, there are two 5 ^' - AATAAA 3 ^' sequences. The SV40-PolyA signal could only be positioned outside the promoters.

Example 2: Singular transfection and analysis of GFP expression from GFP-Bi promoter constructs

The aim of this experiment was to show that both transcripts are produced and that the complementary strands assemble into a dsRNA in a bimolecular reaction. However, it should also be possible to translate the transcript with the regular reading frame if it has not yet been paired with the complementary partner. A reduced rate of GFP protein expression was expected compared to a conventional expression vector due to the competitive assembly of the RNA strands. If transcripts are formed from both promoters, inverting the reading frame of GFP in the bi-promoter construct also leads to a comparable (reduced) expression of GFP.

For this purpose, a complete, an inverted reading frame and a truncated reading frame for GFP were cloned between two promoters and, after transfection, the expression of GFP protein was determined in comparison.

The bi-promoter constructs used here were not generated by gene bank synthesis, but were produced in individual clonings. However, they nevertheless represented constructs that could come from a gene bank synthesis. However, a complete reading frame was cloned here only for test purposes. As the experiments also suggest, it is not aimed at in a gene bank synthesis for bi-promoter constructs. Production of the bi-promoter constructs used:

The vector pcDNA3.1 + (Invitrogen, Karlsruhe) was cleaved with the restriction enzymes Bsml and Smal. Here the neo-resistance gene was removed. The free ends were filled in using Klenow enzyme and religated. The resulting vector was named pcDNA3.1Δneo. This vector was in turn opened with the restriction enzymes Nhel and BamHI in the polylinker sequence and the GFP fragment cut out with the restriction enzymes BglII and Xbal from the vector pEGFP-N2 (GenBank accession number: U57608) was inserted. A CMV promoter fragment from the vector pTet-OFF (GenBank Accession Number: U89929) was inserted into the resulting plasmid via the restriction sites Xhol and EcoRI. The resulting bi-promoter plasmid was named pΔBI-CMV-GFP (Fig. 6).

To construct a vector with an inverted GFP reader, the GFP fragment was cut out from pΔBI-CMV-GFP by NotI and EcoRI cleavage. The ends of all fragments were filled in using Klenow enzyme and the GFP fragment was reinserted. One of the resulting plasmids with the GFP reading frame in the inverted orientation was named pΔBI-CMV-GFP-INV.

Another vector pBI-GFP contained a GFP reading frame shortened at the 5 ^' end without start codon (604 base pairs; from base pair 792 to 1395 relative to GenBank entry U57608). The corresponding sequence was generated by means of PCR from plasmid pEGFP-N2. The primers used (5 ^' primer: 5 ^' - GAATTCGGATCCATGCCACCTACGGCAAGC-3 ^' 3 ^' primer: 5 ^' -

TCTAGAGCGGCCGCTACAGCTCGTCCATGCCG-3 ^' ) also carried cleavage sites for BamHI (5 ^' primer) and Notl (3 ^' primer). After intermediate cloning in pcDNA3.1v5histopo (Invitrogen, Karlsruhe), the PCR fragment was cut out with BamHI and Notl and, instead of the BamHI-Notl fragment, inserted with the complete GFP reading frame in pΔBI-CMV-GFP. The resulting vector was named pBI-GFP.

The vectors pΔBI-CMV-GFP, pΔBI-CMV-GFP-INV, pBI-GFP and the vector pEGFP-N2 were by conventional calcium phosphate transfection in 293 cells transfected and the expression of GFP monitored by fluorescence microscopy 24 hours after the transfection (FIG. 7).

This example shows that the transfection of the bi-promoter constructs with the complete GFP reading frame (A and B) led to a greatly reduced GFP expression in 293 cells compared to the conventional expression plasmid pEGFP-N2 (D). In contrast, the transfection of the bi-promoter plasmid pBI-GFP with 5 ^' -truncated reading frame (C) did not lead to GFP-positive cells. GFP expression in the transfections with the bi-promoter constructs was present, but greatly reduced. Weak GFP expression showed that not all of the transcripts assemble into dsRNA. Some sense transcripts can obviously also be translated. This justifies the need for 5'-truncated cDNAs in gene bank synthesis for bi-promoter constructs, as was demonstrated by the construct pBI-GFP. On the other hand, these GFP-positive cells after transfection of pΔBI-CMV-GFP (A) and pΔBI-CMV-GFP-INV (B) also showed that full-length transcripts were formed by both promoters. This is the evidence for the induced expression of Bi promoter vectors complementary ^RNA's in mammalian cells.

Example 3: Singular transfection and expression analysis of a GFP hairpin construct: extinction of GFP expression

This experiment was intended to determine whether, when expressing a hairpin construct with the complete reading frame of GFP, despite the possibility of intramolecular base pairing to a dsRNA hairpin, GFP protein expression can be detected.

The hairpin construct was cloned by excision of the second promoter from pΔBI-CMV-GFP (see Example 2) using the restriction enzymes EcoRI and Xbal and the insertion of a second GFP reading frame from pEGFP-N2 (GenBank Accession Number: U57608) - which were also cut out using EcoRI and Xbal. As a result, the two reading frames were arranged inverted in the resulting plasmid php-1 (FIG. 8), the first being the anti-sense orientation and then separated by a 29nt long non-complementary sequence the sense orientation of the reading frame came to rest. This arrangement is essential for a third strand synthesis product.

Transfection of php-1 showed no GFP-positive cells, while the conventional GFP expression vector produced numerous GFP-positive transfectants.

It can therefore be assumed that an intramolecular dsRNA formation in the transcript of the hairpin vector presumably results in a masking of the GFP reading frame which is so efficient that translation is no longer possible. When producing a hairpin vector library, in contrast to a bi-promoter library synthesis, there is no need to truncate the reading frames.

Example 4: Use of bi-promoter constructs and hairpin constructs for reducing the gene expression of Firefly luciferase in CGR8 mouse embryonic stem cells

In normal mammalian cell lines, dsRNA causes a general translation stop that is caused by an antiviral mechanism. It has been reported in mouse embryonic cells that this mechanism is not yet active. Therefore, the specific reduction of gene expression by a bi-promoter vector (pBI-Luc) and a hairpin vector (pLuc-hp) in mouse ES cell culture was reproduced, which expresses the gene expression of the transiently coexpressed transcript of the firefly luciferase (photinus pyralis; abbreviated as PP- Luciferase) from the vector pGL3.

The vector pGL3 (GenBank Accession Number: U47296; from Promega, Mannheim) also served as the source for the PP luciferase sequences in the dsRNA vectors. Construction of pBI-Luc and pLuc-hp:

In both vectors, part of the firefly luciferase reading frame - without the start codon - (798 base pairs; from base pair 1131 to 1928 relative to the pGL3 GenBank database entry: U47296) from the expression plasmid pGL3 was used. First of all, this partial sequence was PCR-terminated at the 5 ^' end

Restriction cleavage sites for EcoRI and BamHI and at the 3 ^' end those for Xbal and Notl were added in this order and the product was cloned in a vector for PCR products. (Primers used: 5 ^' primer: 5 ^' -

GAATTCGGATCCCGCTGCTGGTGCCAACCC-3 ^' 3 ^' primer: 5 ^' -

TCTAGAGCGGCCGCACGGCGATCTTTCCGCCC-3 ^' )

The plasmid pBI-Luc was created from pΔBI-CMV-GFP (see patent example 2)

Cut the GFP sequence with the restriction enzymes Notl and BamHI and the insertion of the PP-luciferase-Notl-BamHI-PCR fragment.

The plasmid pLuc-hp was created by cutting out the second promoter from pBI-

Luc using an EcoRI-Xbal cleavage. In its place, the PP luciferase

EcoRI-Xbal-PCR fragment was cloned, so that two firefly-luciferase partial sequences resulted in an inverted arrangement.

The bi-promoter vector pBI-GFP and the hairpin vector php-1 (see patent example 2) with a target sequence different from PP-luciferase served as controls in separate transfection approaches of the same type. It was investigated whether the expressed GFP-specific dsRNA ^it's a nonspecific reducing effect on the PP-luciferase expression have.

To control the transfection efficiency and the undisturbed general gene expression, the expression of Renilla (RL) luciferase was also examined in all transfections - by cotransfection of the vector pRL-Tk (GenBank Accession Number: AF025846; Promega, Mannheim). The transcript of the Renilla luciferase was not sequence homologous to that of the PP luciferase. Each transfection thus comprised three vectors

1. Bi-promoter vector OR hairpin construct OR control vector pcDNA3.1Δneo 2. pGL3 3. pRL-Tk

The vectors were used in the ratio: 5: 1: 2.5 in a transfection. The transfection was carried out by electroporation in mouse CGR8 embryonic stem cells (European Collection of Cell Cultures (ECACC), CAMR, Salisbury, Wiltshire, SP4 OJG, UK; ECACC number: 95011018), which were based on STO-feeder cells (ECACC number: 86032003) in KO-DMEM + 15% serum replacement (Invitrogen, Karksruhe) and with 1000 U / ml LIF factor (from Chemicon, Hofheim) (the Cultivation took place after exercise! et al.) The ES cells were passaged one day before the transfection. Before electroporation, the trypsinized ES and

STO cells plated for at least two hours to pass the STO cells through

Eliminate readhesion from the ES cell suspension. The transfection of 4 E + 6 each

ES cells were carried out in the Easy-Ject-Plus electroporator (from Peqlab, Erlangen) under the following conditions:

Electroporator: 900 μF, 200V. The cells were in simple KO-DMEM

(Invitrogen, Karlsruhe) electroporated, the previously 60 μg herring sperm DNA

(Invitrogen, Karlsruhe), and 10 μg / μl-DEAE-dextran (molecular weight 500,000;

Sigma, Munich) had been added. After electroporation, the

Cells were placed in a 3 cm dish with STO feeder cells and incubated for 24 hours.

All cells were then lysed and the lysates were

Luciferase assay system (Promega, Mannheim) in a Fluoroscan Ascent

Luminescence reader (Labsystems, Frankfurt) the activities of PP and RL

Luciferase (relative light units / second) determined. The PP activities were normalized to the activities of the control gene RL.

Both the bipromotor and the hairpin construct showed a comparable specific reduction in the expression of the Firefly-Luciferase, the constructs pBI-GFP and php-1 which were not specifically directed against the Firefly-Luciferase showed none

Reduction of Firefly Luciferase Expression (Fig. 10).

Example 5: Suppression of the antiviral response in mammalian cell culture by co-expression of the vaccinia virus protein E3L

The aim of this experiment was to co-express the vaccinia virus protein E3L in a transfection with the dsRNA-producing vectors to inhibit the interferon response established in mammalian cells without disturbing the dsRNA-mediated RNAi effect.

The reading frame of the vaccinia virus protein E3L (see GenBank Accession Number NC-001559) is cloned into a eukaryotic expression vector by means of PCR. The resulting expression plasmid was designated pE3L. In the transfection of a bi-promoter vector or one

This expression plasmid is co-transfected into the hairpin vector.

In another embodiment, the E3L expression plasmid may also have been stably introduced into a mammalian cell line beforehand, so that it is expressed either constitutively or under a regulated promoter.

There is a reduction or extinction of the antiviral response (unspecific

Reduction of the total cellular translation machinery) in the mammalian cells, with undisturbed unfolding of the RNAi effect.

Example 6: Enhancement of the RNAi effect in mammalian cell culture by co- / expression of the helicase MOI

By coexpressing the reading frame of the helicase MOl - a putative dsRNA nuclease - (GenBank Accession Number: AB028449) in mammalian cells, two different goals can be achieved: on the one hand, the extent of the interferon response in mammalian cells into which a bi- Promoter or hairpin construct was transfected. This is done by quantitatively reducing the inductor - that is, dsRNA ^'s longer than 30 base pairs - by cleavage before a dsRNA response can be triggered.

On the other hand, in the case of transient transfections of a target sequence, the strength of the RNAi effect can be increased, which in such a case never leads to a complete elimination of the expression of the target sequence, because very large amounts of it are transiently transcribed.

The reading frame of the helicase MOl is cloned into a eukaryotic expression vector in order to obtain the expression plasmid pMOl. When a bi-promoter vector or a hairpin vector is transfected, this expression plasmid pMOl is cotransfected. In another embodiment, the pMOI expression plasmid may also have been stably introduced into a mammalian cell line beforehand, so that it is expressed either constitutively or under a regulated promoter. credentials

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Claims

Expectations

1. Polynucleotide containing an inner polynucleotide which is functionally linked at the 5 ^' end to a first eukaryotic expression control sequence and at the 3 ^' end is functionally linked to a second eukaryotic expression control sequence, wherein

(i) only the first eukaryotic expression control sequence at the 5 'end is functionally linked to a first polyadenylation sequence and the polyadenylation sequence is functional in 3 ^' after 5 ^' orientation, or

(ii) only the second eukaryotic expression control sequence at the 3 ^' end is functionally linked to a second polyadenylation sequence and the polyadenylation sequence is functional in 5 ^' after 3 ^' orientation, or

(iii) the first eukaryotic expression control sequence at the 5 'end is functionally linked to a first polyadenylation sequence and the polyadenylation sequence is functional in 3 ^' after 5 ^' orientation and the second eukaryotic expression control sequence in turn is functionally linked at the 3 ^' end to a second polyadenylation sequence and the polyadenylation sequence is functional in 5 ^' after 3 ^' orientation.

2. The polynucleotide of claim 1, wherein the inner polynucleotide comprises at least 50 nucleotides.

3. The polynucleotide of claim 2, wherein the inner polynucleotide comprises a cDNA molecule or a fragment thereof.

4. The polynucleotide of claim 3, wherein the cDNA molecule or fragment thereof is from a library of cDNA molecules.

A polynucleotide according to any one of claims 1 to 4, wherein the first and second expression control sequences are identical to one another.

The polynucleotide according to any one of claims 1 to 4, wherein the first and second expression control sequences are different from one another.

7. Polynucleotide according to one of claims 1 to 6, wherein the expression control sequence is selected from the group consisting of CMV promoter, thymidine kinase promoter, SV40 promoter, PGK promoter and α-myosin heavy chain promoter.

8. The polynucleotide according to any one of claims 1 to 7, wherein the first and the second expression control sequence are constitutively active.

The polynucleotide according to any one of claims 1 to 6, wherein the first and the second expression control sequence are inducible.

10. The polynucleotide according to claim 9, wherein the inducible first and second expression control sequence is selected from the group consisting of: tetracycline inducible promoters, metallothionine promoters, ecdysone inducible promoters.

11. A polynucleotide according to any one of claims 1 to 10, wherein the first and the second polyadenylation sequence are identical to one another.

12. The polynucleotide of any one of claims 1 to 10, wherein the first and second polyadenylation sequences are different from one another.

13. A method for producing a double-stranded polynucleotide comprising the steps:

(a) Linking a single-stranded first DNA molecule which comprises at the 5 'end a recognition sequence for a restriction endonuclease, at the 3' end of which a second oligonucleotide whose 5 'end is phosphorylated, the second oligonucleotide (i) having a sequence comprising the linkage with the first DNA molecule and (ii) at the 3 'end a sequence of at least 5

Includes nucleotides that form a hairpin

Secondary structure ("stem loop") structure allowed;

(b) Synthesis of a second DNA molecule, the first single-stranded DNA molecule serving as a template for the synthesis of the second DNA molecule and the second DNA molecule serving as a primer starting from the 3 'end of the second oligonucleotide forming a hairpin-shaped secondary structure, at the end the synthesis is a double-stranded DNA molecule consisting of the first DNA molecule, the second oligonucleotide and the second DNA molecule;

(c) denaturing the double-stranded DNA molecule thus obtained; and

(d) Synthesis of a third single-stranded DNA molecule using a third oligonucleotide comprising a sequence identical to the first oligonucleotide, the second single-stranded DNA molecule from step (c) serving as a template and the second and third single-stranded DNA molecules as a double strand at the end of the synthesis.

14. The method of claim 13, wherein the 3 'end of the second oligonucleotide carries a sequence of 3 guanine bases.

15. The method of claim 13 or 14, wherein the single-stranded first DNA molecule is produced by:

(a) Synthesis of a single-stranded first DNA molecule using a first oligonucleotide comprising an oligo-dT sequence and at the δ'-end of the oligo-dT sequence a recognition sequence for a restriction endonuclease, an RNA polyadenylated at its 3'-end A single species molecule serves as a template and the first oligonucleotide is capable of hybridizing to the 3 'end of the polyadenylated RNA molecule;

(b) removing the polyadenylated RNA molecule; and

(c) Provision of the first single-stranded DNA molecule.

16. The method of claim 13, 14 or 15, wherein the polyadenylated RNA

Molecule is obtained by extracting mRNA from cells, tissues or complete organisms or by transcription of cDNA molecules contained in libraries of cDNA molecules.

17. A method for producing a mixture of double-stranded polynucleotides comprising the steps:

(a) Linking of single-stranded first DNA molecules which at the 5 ^' end comprise a recognition sequence for a restriction endonuclease, at the 3 ^' ends of which in each case with a second oligonucleotide whose 5 'end is phosphorylated, the second oligonucleotide (i) comprising a sequence which allows the linkage to the first DNA molecule and (ii) comprises at the 3 ^' end a sequence of at least 5 nucleotides which allows the formation of a hairpin-shaped secondary structure ("stem loop") structure;

(c) denaturing the double-stranded DNA molecule thus obtained; and

18. The method of claim 17, wherein the 3 'end of the second oligonucleotide carries a sequence of 3 guanine bases.

19. The method according to claim 17 or 18, wherein the single-stranded first

DNA molecules are made by:

(a) Synthesis of single-stranded first DNA molecules using first oligonucleotides comprising an oligo-dT sequence and at the 5 'end of the oligo-dT sequence a recognition sequence for a restriction endonuclease, with polyadenylated RNA molecules at their 3' end serve as a template of different species and the first oligonucleotides are able to hybridize with the 3 'ends of the polyadenylated RNA molecules;

(b) removing the polyadenylated RNA molecules; and

(c) Provision of the first single-stranded DNA molecules.

20. The method according to claim 17, 18 or 19, wherein the polyadenylated RNA molecules are obtained by extraction of mRNA from cells, tissues or complete organisms or by transcription of cDNA molecules contained in libraries of cDNA molecules.

21. The method according to any one of claims 13 to 20, wherein the restriction endonuclease is a restriction endonuclease which recognizes a sequence of at least 6 nucleotides.

22. The method according to claim 21, wherein the rarely cleaving restriction endonuclease is selected from the group consisting of: Xho I, Not I, Xba I, Bgl II, Asp 718, Sal I, Sac I, Sfi I.

23. The method according to any one of claims 13 to 22, wherein the sequence from step (a) (i), which allows the linkage, is a 5 'single-stranded region, which serves as the recognition region for the T4 RNA ligase.

24. The method according to any one of claims 13 to 22, wherein the sequence from step (a) (i), which allows the linkage, is a single-stranded 3 'region from 3 to 5 guanine bases, which after hybridization with the 3' region of the single-stranded first DNA molecule is closed by a T4 DNA ligase.

25. The method according to any one of claims 13 to 24, wherein the sequence from step (a) (ii), which allows the formation of a hairpin-shaped secondary structure ("stem loop"), at least 5, 6, 7, 8, 9, 10 or comprises up to 100 nucleotides in length.

26. The method according to any one of claims 13 to 25, wherein in step (d) additionally a fourth oligonucleotide is added, which is phosphorylated on 5 'and comprises a sequence complementary to the second oligonucleotide.

27. The method according to any one of claims 13 to 26, wherein in step (a) the 3 'end of the oligo-dT region of the single-stranded first DNA molecule additionally contains the bases A, C, G.

28. A method for producing a vector or a mixture of vectors, the method comprising the steps of the method according to one of claims 13 to 27 and the additional step of cloning the heterologous polynucleotides produced into a suitable vector.

29. The method of claim 26, 27 or 28, wherein the polynucleotide or the vector is then treated with a T4 (DNA or RNA) ligase.

30. Vector containing a polynucleotide according to one of claims 1 to 12 or a polynucleotide which is produced by a method according to one of claims 13 to 29.

31. Host cell containing the vector according to claim 30 or a vector obtainable by the method according to claim 28.

32. A method for producing a double-stranded RNA which comprises the step of contacting a polynucleotide according to one of claims 1 to 12 or a polynucleotide produced by a method according to one of claims 13 to 29 with a protein or protein mixture under conditions, which allow the synthesis of a double-stranded RNA.

33. The method of claim 32, wherein the protein or protein mixture

Contains T7 polymerase, T3 polymerase or SP6 polymerase.

34. A method for producing a double-stranded RNA, the method comprising the steps of:

(a) introducing a vector according to claim 30 or a vector obtainable by the method according to claim 28 into a host cell; and

35. A method for identifying genes whose inactivation leads to detectable changes in the target cell, the method comprising the steps of the method from claim 34 and the additional step:

Comparison of the phenotype of the host cell from (b) with a host cell into which no vector or a control vector was introduced in step (a).

36. Host cell according to claim 31 or method according to claim 34 or 35, wherein the host cell is a prokaryotic host cell.

37. Host cell or method according to claim 36, wherein the prokaryotic host cell is an E. coli "SURE" cell.

38. The host cell of claim 31 or the method of claim 34 or 35, wherein the host cell is a eukaryotic host cell.

39. Host cell or method according to claim 38, wherein the eukaryotic host cell is selected from the group consisting of 293 cells, NIH3T3 cells, BHK cells, CHO K1 cells, and HeLa cells.

40. Host cell according to claim 31, method according to claim 34 or 35 or host cell or method according to one of claims 36 to 39, wherein in the host cell at least one protein from the group consisting of double-stranded RNA activatable proteins are inactivated or not present in the host cell.

41. Host cell or method according to claim 40, wherein the group of proteins which can be activated by double-stranded RNA comprises protein kinase R (PKR) and RNAse L.

42. Host cell according to claim 31, method according to claim 34 or 35 or host cell or method according to one of claims 36 to 41, wherein the activity of the RNAi-enzyme complex is increased.

43. The host cell or method according to claim 42, wherein the RNAi-enzyme complex has at least one protein which has the biological activity of a protein selected from the group consisting of helicase MOl, nuclease Mut-7 or Dicer.

44. The host cell of claim 31, the method of claim 34 or 35 or the host cell or method of any of claims 36 to 43, wherein the host cell comprises interferon response inhibitory proteins.

45. Host cell or method according to claim 44, wherein the interferon response inhibiting proteins are selected from the group consisting of E1A, HepB virus protein, tetratricopeptide repeat protein, cochaperone p58 (IPK), E3L, or TAR.

46. Transgenic animal containing a polynucleotide according to any one of claims 1 to 12 or a polynucleotide obtainable by a method according to any one of claims 13 to 29.

47. Medicament comprising a polynucleotide according to any one of claims 1 to 12 or a polynucleotide obtainable by a method according to any one of claims 13 to 29.

48. Use of a polynucleotide according to any one of claims 1 to 12 or a polynucleotide obtainable by a method according to any one of claims 13 to 29 for the manufacture of a medicament which can be used for the treatment or prevention of diseases.

49. Use according to claim 48, wherein the disease is selected from the group: cancer, diseases of the cardiovascular system, diseases of the skin, diseases of the internal organs, metabolic disorders, neurological disorders or disorders or disorders or disorders of the immune system, degenerative diseases such as Alzheimer's Disease, Huntington's disease, Parkinson's disease, reperfusion damage, stroke and alcohol damage to the liver, tumor diseases such as leukemia, carcinoma or sarcoma, autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, diabetes lupus, viral diseases such as hepatitis or influenza.