WO2003095652A2 - Produits de recombinaison d'expression utilises pour produire des arn bicatenaires a brin double et leur utilisation - Google Patents

Produits de recombinaison d'expression utilises pour produire des arn bicatenaires a brin double et leur utilisation Download PDF

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WO2003095652A2
WO2003095652A2 PCT/EP2003/004835 EP0304835W WO03095652A2 WO 2003095652 A2 WO2003095652 A2 WO 2003095652A2 EP 0304835 W EP0304835 W EP 0304835W WO 03095652 A2 WO03095652 A2 WO 03095652A2
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sequence
stranded
polynucleotide
host cell
rna
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WO2003095652A3 (fr
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Wolfgang Liebetrau
Dieter Link
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Xantos Biomedicine AG
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Definitions

  • 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
  • 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.
  • 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.
  • 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 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.
  • RNA interference means the nucleolytic cleavage, guided by a double-stranded RNA (dsRNA), of one of the dsRNA sequence homologous mRNAs (Fire et al., 1998).
  • dsRNA double-stranded RNA
  • the specific protein complex required for this must be activated by a dsRNA (Bass, 2000; Carthew, 2001).
  • 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).
  • RNAi is also known in plant genetics under the name PTGS (“post-transcriptional gene silencing”).
  • PTGS post-transcriptional gene silencing
  • 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).
  • RNA-dependent RNA polymerases RdRP
  • RNAi was found in embryos (Yang et al., 2000; Kennerdell and Carthew,
  • RNAi is also effective in mammalian cells.
  • Elbashir et al. 2001
  • 21-nucleotide siRNA small interfering RNAs
  • RNAs 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
  • 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
  • Nonspecific RNAi effects are due, among other things, to the presence of an antiviral common in mammalian cells
  • 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
  • RNA interference RNA interference
  • siRNAs short interfering RNAs
  • RNAi enzyme complex 21-23-mer dsRNA molecules, which arise from processing from longer precursors.
  • 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
  • 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
  • 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.,
  • Target organism are introduced.
  • short dsRNA is also chemically synthesized (Elbashir et al., 2001).
  • C. elegans is a special one
  • Expression plasmid were transformed with oppositely arranged promoters
  • 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
  • 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.
  • 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 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
  • Polyadenylation sequence is functionally linked and the
  • Polyadenylation sequence in 5 'after 3 ' orientation is functional, or
  • 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 '
  • 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.).
  • inner polynucleotide encompasses any polynucleotide which is intended to serve as a template for RNAi molecules.
  • 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
  • cDNAs are particularly preferred, in particular also in
  • 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.
  • 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 include promoter, enhancer and silencer elements.
  • Promoter elements mediate the basal expression of a gene
  • Enhancer elements increase expression, silencer elements, however, reduce or inhibit expression.
  • the promoter, enhancer and silencer elements interact physically with regulatory proteins
  • 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
  • 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 silencing elements negatively interfere with the formation of a complex of the basal transcription factors.
  • 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
  • “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
  • 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.
  • Expression control sequences can be found in known test systems, e.g. through Northern
  • 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 refers to that which occurs after the synthesis of almost all eukaryotic mRNAs at the 3 'end
  • 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
  • the polynucleotide described above containing an inner polynucleotide is used in the
  • dsRNA double-stranded RNA
  • RNAi molecules 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.
  • the polynucleotide according to the invention contains
  • Expression control sequences for example CMV promoters or tk-
  • 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
  • ssRNAs Single stranded RNA molecules in the cell to enable a dsRNA.
  • the promoters should therefore preferably be strong promoters, e.g. CMV
  • 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
  • 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
  • Such an embodiment of the polynucleotide according to the invention is particularly preferred.
  • 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.
  • Polynucleotide should be localized. The reason for this is that dsRNA molecules, which also include expression control sequences, are still unclear
  • strong promoters such as CMV promoters
  • CMV promoters are preferably suitable for the polynucleotides according to the invention.
  • 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 ,
  • 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.
  • 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.
  • HTS high throughput screening
  • RNAi in a screening method has the advantage that a reduction / switch-off of the gene expression of a target gene can be detected.
  • 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.
  • 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).
  • RNA In contrast to inducers for RNAi, antisense molecules, which are mostly used as oligonucleotides, must be present in significantly higher amounts ( ⁇ M 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 also facilitates the introduction of the dsRNA into the cell, since the RNA is expressed in the target cell.
  • 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.
  • the polynucleotides according to the invention can also be used to treat and / or prevent
  • RNAi ds RNA
  • 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.
  • the inner polynucleotide comprises at least 50 nucleotides.
  • the inner polynucleotide comprises a cDNA molecule or a fragment thereof.
  • 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.
  • the cDNA molecule or fragment thereof comes from a library of cDNA molecules.
  • 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.
  • the first and the second expression control sequence are identical to one another or different from one another.
  • the expression control sequence is selected from the group consisting of CMV promoter, thymidine kinase promoter, SV40 promoter or PGK promoter, ⁇ -myosin heavy chain promoter.
  • the first and the second expression control sequence are constitutively active.
  • constitutitutively active expression control sequence denotes an expression control sequence which is activated by the transcription machinery already contained in the cell.
  • the first and the second expression control sequence are inducible.
  • 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.
  • 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.
  • 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, Düsseldorf).
  • 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).
  • the first and the second polyadenylation sequence are identical to one another.
  • 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:
  • step (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
  • production also includes additional steps, such as pretreatments of the starting material or
  • 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
  • restriction endonuclease recognition sequence includes one
  • Restriction endonuclease can range from 4 to 10 base pairs. As part of the
  • recognition sequences are preferred which comprise 6 base pairs and more.
  • 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.
  • 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.
  • 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 '
  • 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
  • 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.
  • 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.
  • the hairpin loop forms after hybridization of the 3 'end of the oligonucleotide with its 5' end.
  • 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.
  • synthesis encompasses the linking of nucleotides to polynucleotides.
  • 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.)
  • Polynucleotide triggers Denaturation is also known as melting.
  • Denaturation of polynucleotides can be achieved, for example, by increasing the rate
  • 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.
  • third oligonucleotide includes an oligonucleotide that is one of the first
  • Oligonucleotide has identical sequence. With the first oligonucleotide in
  • 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
  • Hairpin structure binds to avoid refolding of the second strand product.
  • 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
  • (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
  • Second strand synthesis performed.
  • the result is an uninterrupted, unilaterally covalently closed paired DNA second strand synthesis product.
  • 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.
  • the third-strand synthesis product should still be treated with T4 DNA ligase before it is cloned into a common expression plasmid.
  • T4 DNA ligase Such a construct is also called as
  • Hairpin expression vector (see for example Figure 2).
  • 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.
  • 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
  • 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 The two strands of the RNAi molecule.
  • the single-stranded first DNA molecule of the method according to the invention is produced by:
  • 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-
  • polyadenylated RNA molecule of a single species means one or more identical mRNA molecules. This includes mRNA
  • MRNAs are preferred
  • mRNAs Mammals, especially human mRNAs. mRNAs can be made up of cells,
  • Body fluids such as lymph, serum, plasma, urine, spinal fluid etc.
  • hybridization means within the scope of this invention
  • Hybridization under conventional hybridization conditions preferably under stringent conditions, as described, for example, in Sambrook (Molecular
  • the term "remove” includes the separation and removal of the building blocks of the polyadenylated RNA molecule.
  • the mRNA molecules can be removed by incubation with RNases or by alkaline hydrolysis. Incubation with RNase H is preferred.
  • supply includes purification methods and
  • 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.
  • the invention further relates to a method for producing a mixture of double-stranded polynucleotides comprising the steps:
  • step (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.
  • mixture of double-stranded polynucleotides denotes a multiplicity of double-stranded polynucleotides according to the invention, which comprise identical or different nucleic acid molecules.
  • the single-stranded first DNA molecules of the method according to the invention are produced by:
  • 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.
  • 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.
  • the restriction endonuclease recognizes a sequence of at least 6 nucleotides.
  • 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.
  • 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.
  • 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.
  • 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.
  • “Stem loop”) allows at least 5, 6, 7, 8, 9, 10 or up to 100 nucleotides in length.
  • a fourth oligonucleotide is added in step (d), which is phosphorylated on 5 ' and comprises a sequence complementary to the second oligonucleotide.
  • 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.
  • vector refers to prokaryotic or eukaryotic cloning and / or expression vectors.
  • 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 vectors can be the same or different according to the invention.
  • 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.
  • heterologous polynucleotides means in the context of
  • Polynucleotides of the invention can be from different species.
  • 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.
  • the invention relates to a host cell which contains a vector 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
  • 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.
  • 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.
  • the term “protein or protein mixture” means a protein or protein mixture which is able to catalyze the synthesis of RNA molecules.
  • proteins are preferably RNA polymerases. Suitable RNA polymerases are described in more detail below.
  • 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.
  • the protein or protein mixture contains T7 polymerase, T3 polymerase or SP6 polymerase.
  • T7 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:
  • introduction encompasses all types of physical or chemical interactions between the polynucleotides and the cell or the cellular components.
  • 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.
  • a suitable liquid for example a nutrient medium for the cell
  • gels and gel-like liquids can also be used.
  • introduction also optionally includes the integration of the polynucleotide into the genome of the host cell.
  • nucleic acids examples include 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.
  • 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
  • a culture medium which contains nutrients and, if appropriate, growth and
  • Vectors containing the polynucleotide of the invention can be obtained via conventional transfection methods such as e.g. by calcium phosphate transfection
  • Electroporation, viral transfer or other transfection methods into which cells are introduced The cultivation of these cells under conditions that a
  • 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.
  • 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.
  • 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.
  • Cell morphology can e.g. be examined by means of morphometric methods.
  • activation 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
  • 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
  • phenotype denotes the appearance of a cell that is characterized by the
  • Phenotype includes all external and internal structures and functions of the cell.
  • control vector denotes the vector used for the above-mentioned method, which, in contrast to this, is not an inventive one
  • Polynucleotides or vectors can be checked or ensured by suitable controls in which target cells are transfected, for example with this control vector.
  • 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.
  • target cells in the context of the invention includes eukaryotic cells
  • Cells especially mammalian cells and preferably human cells, in which specifically suppress or at least reduce the expression of a target gene.
  • the host cell is a prokaryotic host cell.
  • the prokaryotic host cell is an E. coli “SURE” cell.
  • the host cell is a eukaryotic host cell.
  • the eukaryotic host cell is selected from the group consisting of 293 cells, NIH3T3 cells, BHK cells, CHO K1 cells, and HeLa cells.
  • 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.
  • 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.
  • PKR 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
  • PLR antiviral response
  • PKR antiviral response
  • 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).
  • RNA interference the initiating double-stranded RNAs are first broken down into short interfering RNAs (siRNAs).
  • siRNAs 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:
  • 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.
  • RNAi-enzyme complex a cell line in which inhibition of the interferon response has been described.
  • 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:
  • RNA 's 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).
  • the group of proteins which can be activated by double-stranded RNA comprises protein kinase R (PKR) and RNAse L (loc. Cit).
  • the activity of the RNAi-enzyme complex is increased.
  • 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.
  • 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.).
  • the host cell comprises proteins that inhibit the interferon response.
  • interferon response encompasses an antiviral mechanism which is widespread in mammalian cells and which can be attributed to non-specific RNAi effects.
  • 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.
  • 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.
  • transgenic mice 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
  • transgenic animals according to the invention can be used as a model for
  • 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.
  • 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.
  • 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.
  • Suitable pharmaceutically acceptable carriers 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 ⁇ g 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
  • 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.
  • treatment here denotes therapeutic measures for combating, inhibiting, eliminating or alleviating diseases
  • 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,
  • Invention can be transferred into cells by non-viral gene therapy techniques, including receptor-mediated, targeted DNA transfer using
  • 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.
  • 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.
  • a pharmaceutically acceptable carrier excipient and / or diluent.
  • 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
  • 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
  • the flow chart shows the individual steps in the construction of a hairpin expression vector
  • 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).
  • 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.
  • the construct obtained in this way can be inserted into a suitable expression vector regardless of the orientation (6).
  • the antisense sequence appears in the
  • Fig. 4 schematic representation of the vector ptwopA
  • 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.
  • 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).
  • Reading frame (C) did not lead to GFP-positive cells.
  • these GFP-positive cells showed after transfection of p ⁇ BI-CMV-GFP (A) and p ⁇ BI-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
  • 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 (pcDNA3.1 ⁇ neo).
  • 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.
  • 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.
  • GFP green fluorescent protein
  • 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.
  • 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.
  • 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.
  • 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, Düsseldorf) 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 pcDNA3.1 ⁇ neo. 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 p ⁇ BI-CMV-GFP (Fig. 6).
  • the GFP fragment was cut out from p ⁇ BI-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 p ⁇ BI-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).
  • BamHI 5 ' primer
  • Notl 3 ' primer
  • 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 p ⁇ BI-CMV-GFP.
  • the resulting vector was named pBI-GFP.
  • the vectors p ⁇ BI-CMV-GFP, p ⁇ BI-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).
  • 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 p ⁇ BI-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.
  • 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.
  • Example 4 Use of bi-promoter constructs and hairpin constructs for reducing the gene expression of Firefly luciferase in CGR8 mouse embryonic stem cells
  • 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.
  • pBI-Luc bi-promoter vector
  • pLuc-hp hairpin vector
  • 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:
  • the plasmid pBI-Luc was created from p ⁇ BI-CMV-GFP (see patent example 2)
  • the plasmid pLuc-hp was created by cutting out the second promoter from pBI-
  • the bi-promoter vector pBI-GFP and the hairpin vector php-1 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.
  • 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.
  • Bi-promoter vector OR hairpin construct OR control vector pcDNA3.1 ⁇ neo 2.
  • pGL3 3.
  • 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
  • ES cells were carried out in the Easy-Ject-Plus electroporator (from Peqlab, Erberg) under the following conditions:
  • Electroporator 900 ⁇ F, 200V. The cells were in simple KO-DMEM
  • 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.
  • 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.
  • pE3L In the transfection of a bi-promoter vector or one
  • This expression plasmid is co-transfected into the hairpin vector.
  • 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.
  • Example 6 Enhancement of the RNAi effect in mammalian cell culture by co- / expression of the helicase MOI
  • dsRNA nuclease - 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.
  • the reading frame of the helicase MOl is cloned into a eukaryotic expression vector in order to obtain the expression plasmid pMOl.
  • this expression plasmid pMOl is cotransfected.
  • 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
  • Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev., 15 (20): 2654-9.
  • dsRNA-mediated gene silencing in cultured Drosophila cells a tissue culture model for the analysis of RNA interference. Gene., 252 (1-2): 95-105.
  • RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus.
  • RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev., 15 (2): 188-200.
  • VAI RNA virus-associated RNA I
  • H1.1 histone H1 isoform
  • Adenovirus VAI RNA antagonizes the RNA editing activity of the ADAR adenosine deaminase. Virology., 245 (2): 188-96.
  • TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR. Proc Natl Acad Sei U SA., 91 (11): 4713-7.
  • Vaccinia virus E3L protein is an inhibitor of the interferon (i.f.n.) - induced 2-5A synthetase enzyme. Virology., 243 (2): 406-14.
  • RNA the transcript from a master gene for ID element amplification, is able to prime its own reverse transcription.
  • Double-stranded RNA is a trigger for apoptosis in vaccinia virus-infected cells. J Virol., 71 (3): 1992-2003.
  • Adenovirus VA RNAI a positive regulator of mRNA translation. Mol Cell Biol., 4 (4): 736-42.
  • Zamanian-Daryoush M., Der, S.D., Williams, B.R., (1999). Cell cycle regulation of the double stranded RNA activated protein kinase, PKR. Oncogene., 18 (2): 315-26.
  • RNAi double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell., 101 (1): 25-33.

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Abstract

L'invention concerne un polynucléotide contenant un polynucléotide intérieur, lié de manière fonctionnelle en terminaison 5' à une première séquence de contrôle d'expression eucaryote et, en terminaison 3', à une seconde séquence de contrôle d'expression eucaryote, également de manière fonctionnelle. (i) seule la première séquence de contrôle d'expression eucaryote est liée de manière fonctionnelle en terminaison 5' à une première séquence de polyadénylation et ladite séquence de polyadénylation est fonctionnelle en 3' en fonction de l'orientation de 5', ou (ii) seule la seconde séquence de contrôle d'expression eucaryote est liée de manière fonctionnelle en terminaison 3' à une seconde séquence de polyadénylation et ladite séquence de polyadénylation est fonctionnelle en 5' en fonction de l'orientation de 3', ou (iii) la première séquence de contrôle d'expression eucaryote est liée de manière fonctionnelle en terminaison 5' à une première séquence de polyadénylation et ladite séquence de polyadénylation est fonctionnelle en terminaison 3' en fonction de l'orientation de 5' et la seconde séquence de contrôle d'expression eucaryote est liée quant à elle en terminaison 3' de manière fonctionnelle à une seconde séquence de polyadénylation et ladite séquence de polyadénylation est liée de manière fonctionnelle en terminaison 5' en fonction de l'orientation de 3'. L'invention concerne en outre des procédés permettant de produire des polynucléotides à brin double. L'invention concerne également des vecteurs et des mélanges de vecteurs, ainsi que des procédés permettant de produire des vecteurs qui comprennent les polynucléotides selon l'invention ou les polynucléotides produits selon les procédés de l'invention, ainsi que des cellules hôtes qui contiennent ces vecteurs. L'invention concerne en outre des procédés permettant d'identifier des gènes, dont l'inactivation induit des modifications décelables d'une cellule cible. L'invention concerne par ailleurs des animaux transgéniques comprenant un polynucléotide selon l'invention. Pour finir, l'invention concerne l'utilisation du polynucléotide selon l'invention pour produire un médicament pour traiter ou prévenir des maladies.
PCT/EP2003/004835 2002-05-08 2003-05-08 Produits de recombinaison d'expression utilises pour produire des arn bicatenaires a brin double et leur utilisation Ceased WO2003095652A2 (fr)

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DE102004010547A1 (de) * 2004-03-03 2005-11-17 Beiersdorf Ag Oligoribonukleotide zur Behandlung von irritativen und/oder entzündlichen Hauterscheinungen durch RNA-Interferenz

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WO1997004131A1 (fr) * 1995-07-21 1997-02-06 Forsyth Dental Infirmary For Children Amplification d'epingles a cheveux de polynucleotide avec une seule amorce
US6087099A (en) * 1997-09-08 2000-07-11 Myriad Genetics, Inc. Method for sequencing both strands of a double stranded DNA in a single sequencing reaction
GB9827152D0 (en) * 1998-07-03 1999-02-03 Devgen Nv Characterisation of gene function using double stranded rna inhibition
AU2368201A (en) * 1999-12-23 2001-07-09 Xantos Biomedicine Ag Screening method for nucleic acids
EP1272630A2 (fr) * 2000-03-16 2003-01-08 Genetica, Inc. Procedes et compositions d'interference d'arn
GB0012233D0 (en) * 2000-05-19 2000-07-12 Devgen Nv Vector constructs
WO2001092513A1 (fr) * 2000-05-30 2001-12-06 Johnson & Johnson Research Pty Limited Methodes permettant d'agir sur la suppression de gene par utilisation de facteurs renforçant l'arni
US20030198627A1 (en) * 2001-09-01 2003-10-23 Gert-Jan Arts siRNA knockout assay method and constructs

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