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WO2003076668A2 - Procede d'identification d'inhibiteurs d'arn specifiques - Google Patents

Procede d'identification d'inhibiteurs d'arn specifiques Download PDF

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Publication number
WO2003076668A2
WO2003076668A2 PCT/EP2003/002350 EP0302350W WO03076668A2 WO 2003076668 A2 WO2003076668 A2 WO 2003076668A2 EP 0302350 W EP0302350 W EP 0302350W WO 03076668 A2 WO03076668 A2 WO 03076668A2
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Prior art keywords
nucleic acid
toxin
cell
rnase
specific rna
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PCT/EP2003/002350
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German (de)
English (en)
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WO2003076668A3 (fr
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Martin Rohrbach
Martin Funk
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Medigene Ag
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Priority to AU2003214103A priority Critical patent/AU2003214103A1/en
Publication of WO2003076668A2 publication Critical patent/WO2003076668A2/fr
Publication of WO2003076668A3 publication Critical patent/WO2003076668A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the invention relates to a method for identifying substances which are suitable for specifically inhibiting the expression of a protein or the action of a nucleic acid.
  • the invention further relates to an RNA coding for a toxin and a target protein or a target nucleic acid, and to the use thereof for identifying specific RNA inhibitors.
  • RNA an RNA
  • RNA inhibitors which are suitable for specifically inhibiting the expression of a protein starting from an RNA (usually an mRNA) or which are suitable for specifically inhibiting the action of RNAs (specific RNA inhibitors) can be used directly as therapeutic agents.
  • the effect of such specific RNA inhibitors is based on the sequence-specific interaction with the RNA coding for the protein or the nucleic acid.
  • the structure and / or the function of coding RNA sequences is changed such that the expression of the protein originally encoded by the RNA or the action of the RNA is inhibited or completely prevented.
  • the sequence-specific interaction mostly leads to an enzymatic degradation of the RNA (Branch AD: A hitchhikers guide to antisense and non-antisense biochemical pathways. Hepatology 1996; 24: 1517-1529). Since the mRNA in the cell's cytosol is generally freely accessible, inhibiting the mRNA is easier than blocking the DNA coding for the mRNA in the cell nucleus.
  • RNA inhibitors known in the art include, for example, antisense oligonucleotides, ribozymes or dsRNAs.
  • antisense oligonucleotides are understood to mean short-chain oligonucleotides which are linked via complementary base pairing bind corresponding sequences of the RNA and thereby inhibit the expression of proteins or nucleic acids.
  • the antisense oligonucleotide can be a DNA, an RNA or a modified nucleic acid.
  • Antisense oligonucleotides can be expressed intracellularly, or they can also be produced synthetically and modified in such a way that sufficient stability against degrading enzymes is ensured. The most important of these modifications is the so-called phosphorothioate modification. An oxygen atom in the phosphate is replaced by a sulfur atom. In addition, however, a methoxy modification or a 2 '-5' phosphate linkage is also possible (Usmann N. and Blatt LM; J. Clin. H vest. 2000 Nov; 106 (10): 1197-202).
  • Double-stranded RNA can also be used as an RNA inhibitor.
  • dsRNAs denotes naturally occurring or synthetic produced double-stranded RNA sequences which initiate an interaction with cellular RNA sequences and ultimately cause their degradation.
  • a strand of the dsRNA must be complementary to the sequence of the RNA to be inhibited. The effect of the dsRNA is probably due to the fact that the dsRNA forms a triple helix together with the RNA to be inhibited, which is then broken down in the cell.
  • dsRNA which is specifically active in mammalian cells, is not possible using methods known in the art (Elbashir et al. Nature 2001; 411: 494-498).
  • Short dsRNA leads to sequence-specific RNA interference in the mammalian cell, which means that only the RNA to be inhibited is degraded.
  • longer dsRNA at least 30 bp generally causes the unspecific degradation of all RNA and the inhibition of protein synthesis (Hunter et al. 1975, J Biol Chem 250; 409-417).
  • sequence-specific RNA interference thus describes a process in which the introduction of double-stranded RNA sequences into a cell results in a sequence-specific inhibition of gene expression.
  • the dsRNA is first broken down into short sequences of 21-23 bp, so-called siR As (short interference RNAs) These siR As then use cellular enzymes to break down the mRNA that is homologous to the respective siRNA.
  • ribozymes composed of ribonucleic acids (RNAs) which are able to specifically cleave phosphodiester bonds specifically and in some cases also to link them. So that ribozymes can be used to be specific To bind RNA (mostly mRNA) specifically by complementary base pairing and then cleave it using its catalytic activity. This prevents the translation of the (m) RNA into the corresponding protein or inhibits the action of the RNA.
  • RNAs ribonucleic acids
  • WO 95/29254 describes an in vivo method in the yeast Schizosaccharomyces pombe for screening for effective antisense oligonucleotides which are intended to inhibit the expression of target proteins.
  • the gene coding for the target protein is fused to a reporter gene.
  • Antibiotic resistance genes, resistance genes against chemical substances, fluorescent proteins, essential growth factors and other reporter genes are mentioned as possible fusion partners.
  • the aim is to identify antisense oligonucleotides which are intended to inhibit the expression of the target protein and thus of the reporter gene.
  • all of the reporter genes disclosed in WO 95/29254 give the cell an advantage (for example resistance to antibiotics) or enable the cell to be identified (for example by forming a fluorescent protein). The result of this is that cells which contain an effective antisense oligonucleotide either die or cannot be identified, with the disadvantage that RNA can no longer be isolated from these cells.
  • RNA inhibitors accordingly leads to a negative result in the method described in WO 95/29254.
  • a negative detection is unsuitable for the reliable identification of inhibitors, since the sensitivity of the test system is often insufficient to distinguish between positive and negative results. If, for example, a reporter gene is only expressed weakly anyway, inhibitor must be added - and thus an even further decreasing expression - often no clear statements about a successful inhibition possible.
  • Another disadvantage of this screening system is that the loss of expression of a reporter gene could not only be due to the action of the RNA inhibitor.
  • the cause could also be the influencing of other cellular processes, such as an effect of the potential inhibitor on proteins which are involved in translation, translocation and protein processing. This effect could also indirectly influence the expression of the reporter protein.
  • the inhibitor can also act directly on replication mechanisms and the cell cycle and thus inhibit cell growth or generally have a cytotoxic effect.
  • a detection method that is based on a negative detection basically has a lack of specificity due to the unclear location of the inhibitor.
  • the object of the present invention is therefore to provide a method with which specific RNA inhibitors can be identified efficiently. It is also an object of the present invention to provide substances which can be used in the process according to the invention.
  • the object is achieved by a method for identifying specific RNA inhibitors, characterized by the steps: a) introducing at least one type of nucleic acid (fusion gene) coding for at least one toxin and for at least one target protein, a target nucleic acid or for parts of at least one target protein or at least one target nucleic acid, it being possible for an RNA to be formed from the fusion gene, which encoded both for the toxin and for the target protein or the target nucleic acid or parts thereof (mRNAl); b) introducing one or more specific RNA inhibitors into the cell, steps a) and b) being able to be carried out in any order or simultaneously; c) incubating the cell under conditions that
  • the present invention thus provides for the first time a method in which the effectiveness of potential RNA inhibitors is determined on the basis of their ability to inhibit the expression of a toxin fused to the coding region of the target protein or the target nucleic acid.
  • the method according to the invention is therefore based on the principle of positive selection: hl cells which contain effective RNA inhibitors, the toxin cannot develop its action, since these inhibit the expression of the toxin. These cells can survive and grow, whereas cells that do not contain effective RNA inhibitors die off or their growth is drastically inhibited.
  • the method according to the invention has the advantage that the molecules involved are present in the same form, ie with correct folding and the same modification and or interaction with cellular factors, as in the mammalian cell to be treated later. gene and that the in vivo situation of the use of specific RNA inhibitors is correctly reflected.
  • the present invention thus makes it possible to specifically identify effective RNA inhibitors, in particular antisense oligonucleotides, ribozymes or dsRNAs, in a universal in vivo method. It is particularly advantageous that several substances for a rough screening can first be tested simultaneously before individual suitable molecules are identified. In addition, due to the positive selection principle, the method can also be carried out in a multiplex setup for several targets. Another decisive advantage of the method according to the invention is that it is possible to isolate and identify the active RNA inhibitors from the cells.
  • RNA inhibitors are understood to mean substances which change the structure and / or function of coding RNA sequences in such a way that the expression of the protein coded by the RNA or the action of the RNA is inhibited or completely is prevented.
  • the specific RNA inhibitors are selected from the group consisting of antisense OUgonucleotides, ribozymes and dsRNA.
  • molecules comprising antisense oligonucleotides fused with ribozymes or tRNA or other RNA species fused with ribozymes can also be tested within the scope of the method according to the invention.
  • At least one type of nucleic acid is introduced into a cell which is responsible for at least one toxin and at least at least one target protein or a target nucleic acid or parts thereof.
  • This nucleic acid is referred to below as the fusion gene.
  • 1 to 5 different, particularly preferably 1 to 3, and very particularly preferably 1 fusion gene are preferably introduced into the cell.
  • the fusion gene preferably codes for 1 to 5 toxins, preferably for 1 to 3 toxins and very particularly preferably for 1 toxin.
  • the fusion gene preferably codes for 1 to 5, in particular 1 to 3, target proteins, target nucleic acids or for parts of 1 to 5, in particular 1 to 3, target proteins or target nucleic acids.
  • the fusion gene very particularly preferably codes for a target protein, a target nucleic acid or for parts of a target protein or a target nucleic acid.
  • the toxin encoded by the fusion gene includes any toxin which is capable of inhibiting or weakening a function of the cell.
  • Such functions include, for example, cell proliferation, cell growth, or the chemical conversion of molecules such as the cleavage of molecules.
  • toxins are known in the prior art which can be used in the process according to the invention, such as, for example, diphtheria toxin, cholera toxin, pertussis toxin, enterotoxin, botulinum toxin C2, botulinum toxin C3, shiga toxin, verotoxins, tetanus toxin, RNase T1, RNase JH ,
  • the toxin inhibits or weakens cell proliferation. It also includes those toxins that kill the cells.
  • the fusion gene codes for a reversible toxin.
  • the term “reversible toxin” or “toxin with reversible action” denotes a toxin which inhibits the proliferation of cells efficiently and in the long term without killing or seriously damaging the cell. The effect of the toxin can thus be abolished again, so that after the expression of the toxin ceases, the cells start to proliferate again rapidly.
  • the reversible toxin encoded by the fusion gene contains at least two nucleases.
  • nucleases refer to proteins with nuclease activity, specifically both exonucleases and endonucleases, in particular restriction endonucleases.
  • Naturally occurring nucleases which are suitable for use as a reversible toxin according to the invention are known, for example, from bacteria, fungi and mammals. These nucleases include DNAsel, EcoRI, RNAse Tl, RNAse III (Wang X. et al. Nucleic Acids Res. (2001) 29, 1643-1950; Barnes G. et al. Proc. Natl. Acad. Sei. USA (1985) 82, 1354 -8; Fujii, T. et al. Biosci. Biotechnol Biochem. (1995) 59, 1869-74).
  • nuclease also includes functional variants of naturally occurring nucleases which also show nuclease activity.
  • This can be, for example, a protein with a protein sequence which is homologous to that of natural nucleases occurring in bacteria, fungi and mammals, the homology at the amino acid level being at least about 30%, preferably at least about 50%, at least about 60%, is at least about 70% or at least about 80% and is particularly preferably at least about 90%.
  • nucleases also includes functional variants with nuclease activity which, in comparison to naturally occurring nucleases, contain one or more mutations, insertions and / or deletions of individual or more amino acids.
  • Deletions according to the invention preferably comprise 1-50 amino acids, in particular 1-20 amino acids and particularly more preferably 1-10 amino acids.
  • the suitability of a nuclease for use in the context of a reversible toxin can easily be determined by the expression of the nuclease under the control of an inducible promoter.
  • the test of the reversible toxin is suitable, for example, when using S. cerevisiae the MET25 or GALl promoter and when using mammalian cells a tetracycline inducible promoter.
  • a suitable functional variant of a nuclease shows, for example, expression under the control of a suitable inducible promoter at least about 20%, at least about 50%, at least about 100%> and particularly preferably at least about 200% of the inhibition of cell growth of the natural nuclease from which it is derived is.
  • the at least two nucleases are linked to one another covalently or non-covalently.
  • the connection can take place via any amino acid residue of the nucleases. Linking the N- and N-terminus, C- and N-terminus and / or C- and C-terminus of the respective RNases is particularly preferred. Examples of covalent or non-covalent linking of proteins are known to the person skilled in the art (Holst HU et al., Eur. J. Hum. Genet.
  • the nucleases can be connected to one another directly or via a linker.
  • the nucleases are covalently bound to one another via a linker.
  • a linker comprises an amino acid chain of approximately 1 to approximately 50 amino acids in length.
  • Such a linker has the task, for example, of spatially separating the nucleases or of allowing the fusion protein to be correctly folded from the at least two nucleases. Therefore, in a preferred embodiment, the linker primarily comprises the linker in a preferred embodiment primarily alanine residues, which ensures great flexibility of the linker connection existing between the fusion proteins.
  • the reversible toxin contains at least two DNAses, at least two RNAses or at least one DNAses and at least one RNAses.
  • the reversible toxin contains only DNAsen or RNAsen as nucleases.
  • the reversible toxin contains RNase T1 and RNase III (Nicholson, AW, supra) and / or functional variants thereof, the RNase T1 (Fujii, T. et al., Supra) preferably from Aspergillus oryzae and the RNase III preferably from Escherichia coli.
  • a reversible toxin which contains the RNAse T1 and / or functional variants thereof N-terminal and the RNAse III and / or functional variants thereof C-terminal can inhibit the growth of cells particularly efficiently.
  • the invention therefore preferably relates to a reversible toxin in which the RNAase T1 and / or functional variants thereof are N-terminal and the RNAse III and / or functional variants thereof are C-terminally arranged on a polypeptide.
  • the reversible toxin contains, as a further component, a protein without nuclease activity, in particular proteins such as proteases, pore-forming enzymes, and / or modifying enzymes (such as, for example, ADP-ribosylating enzymes (for example pertussis or cholera toxin), glycosylating and / or phosphorylating enzymes).
  • proteins such as proteases, pore-forming enzymes, and / or modifying enzymes (such as, for example, ADP-ribosylating enzymes (for example pertussis or cholera toxin), glycosylating and / or phosphorylating enzymes).
  • the reversible toxin contains, as a further component, a substance which affects the sensitivity of cells to increased above the nuclease activity, in particular the RNAse activity. This is particularly advantageous in cases where the nuclease effect is only weak.
  • the fusion gene codes for a target protein and / or a target nucleic acid or for parts thereof.
  • target protein is to be understood as a protein whose expression is to be inhibited by the action of the specific RNA inhibitor.
  • target nucleic acid is to be understood as an RNA and the effect thereof to be inhibited by the action of the specific RNA inhibitor.
  • the fusion gene codes for parts of the target protein or the target nucleic acid.
  • the part of the target protein has a length of at least 5 amino acids, preferably 10 amino acids and particularly preferably 15 amino acids.
  • the part of the target nucleic acid has a length of at least 15 nucleic acids, preferably 30 nucleic acids and particularly preferably 45 nucleic acids.
  • the nucleic acid segments coding for the toxin and for the target protein and / or the target nucleic acid can be arranged as desired in the fusion gene.
  • the nucleic acid coding for the target protein and / or the target nucleic acid are arranged N-terminally and the nucleic acid coding for the toxin C-terminally on the fusion gene.
  • the fusion gene additionally contains a reporter gene.
  • a reporter gene can be used to ensure that the cells express the toxin.
  • a re- portergen understood a gene that codes for a protein, the expression of which can be detected by suitable tests or in some other way.
  • An example of this is a fluorescent protein (e.g. GFP) (Chalfie, M., et al., Green Fluorescent Protein as a Marker for Gene Expression "Science, 263: 802 (1994); van Roessel, P. et al, Nat. Cell. Biol.
  • Suitable reporter genes include, but are not limited to, BFP, CFP, GFP, YFP, His3, CAT, GUS LacZ, or luciferase (Welsh S. et al., Curr. Opin. Biotechnol. 1997 Oct; 8) 5): 617- 22; Naylor L.H., Biochem. Pharmacol. 1999 Sept 1; 58 (5): 749-57; Schenborn E. et al., Mol. Biotechnol. 1999 Nov; 13 (1): 29-44).
  • the fusion gene is part of a vector.
  • Vector is understood to mean any DNA (possibly combined with other substances such as proteins) into which the fusion gene can be ligated and which is used to introduce and / or multiply the DNA in a cell.
  • the vector is a plasmid or a virus, in particular a recombinant adenovirus.
  • Suitable plasmids and vectors are known to the person skilled in the art (Altschuh et al., Current Protokols in Molecular Biology; Molecular Cell Biology, Lodish H. et al., Fourth Edition WH Freeman; Presutti C. and Santoro B .; Ann Ist Super Sanity 1991; 27 (1): 105-14; Benihoud K. et al.,; Curr. Opion. Biotechnol. 1999 Oct; 10 (5): 440-7; Kovesdi I. et al .; Curr. Opion. Biotechnol. 1997 Oct ; 8 (5): 583-9).
  • the fusion gene is introduced into a cell.
  • Any eukaryotic cell is suitable as a cell, in which, starting from the fusion gene, an RNA can be formed which contains both the toxin and the target protein or encodes the target nucleic acid or parts thereof (mRNAl) and in which the toxin can be expressed.
  • the cell is a yeast cell, preferably S. cerevisae; according to a further preferred embodiment, the cell comes from an immortalized cell line, in particular a mammalian cell line.
  • the cell can also originate from a primary culture of eukaryotic cells, in particular mammalian cells. Suitable yeast cells, eukaryotic cells or immortalized cell lines are known to the person skilled in the art. (Molecular Biology of the Cell, 3 rd edn., 1994, Alberts at al.)
  • the cell is a heart cell.
  • the cell comes from a cell line selected from the group consisting of HELA cells, COS cells, CHO cells, 3T3 cells, L cells, Jurkat cells, BHK cells and HEK cells ( American Type Culture Collection (ATTC) catalog, Manassas, VA, USA).
  • the fusion gene can be introduced into the cell in any way known to the person skilled in the art (Altschuh et al, supra). Such methods include, for example, lipofection, electroporation, or calcium phosphate-mediated transfection.
  • the specific RNA inhibitor is introduced into the cell by introducing a nucleic acid coding for it into the cell.
  • this nucleic acid can be a DNA or an RNA, preferably it is a DNA.
  • the nucleic acid coding for the specific RNA inhibitor is part of a vector.
  • the vector which contains the nucleic acid coding for the specific RNA inhibitor additionally contains a reporter gene.
  • the reporter gene reference is made to the above explanations regarding reporter genes.
  • the preferred embodiments are as defined above.
  • the reporter gene which is contained in the vector containing the fusion gene is different from the reporter gene which is contained in the vector which contains the nucleic acid coding for the specific RNA inhibitor. This enables the expression of the specific RNA inhibitor to be controlled independently of the formation of the mRNAl and the expression of the toxin.
  • steps a) and b) according to the invention can be carried out simultaneously, d. H. the fusion gene and the potential specific RNA inhibitors and / or the nucleic acids coding therefor are simultaneously introduced into the cell. This ensures that the specific RNA inhibitors can develop their effect before the toxin is even expressed, so that damage is avoided from the outset in the case of effective specific RNA inhibitors.
  • step b) is carried out before step a).
  • this embodiment prevents the cell from being damaged by the expression of the toxin before the specific RNA inhibitors can take effect.
  • step a) is carried out before step b).
  • This embodiment is particularly preferred when a toxin with a reversible effect is used as the toxin and when it is intended to test many potential specific RNA inhibitors at the same time.
  • This embodiment makes it possible to prepare a test system which onsgen containing cells, into which the individual potential specific RNA inhibitors can then be introduced.
  • the cell is cultivated under conditions which enable the formation of the mRNAl and which are suitable for the expression of the toxin on the basis of the mRNAl.
  • Such conditions depend on the particular fusion gene and are known to the person skilled in the art.
  • the formation of the mRNAl is controlled by constitutive or regulatable promoters.
  • the expression of the specific PJSfA inhibitor is controlled by constitutive or regulatable promoters.
  • control of the formation of the mRNAl and the control of the expression of the specific RNA inhibitor can be carried out jointly or independently of one another.
  • Such promoters are known to the person skilled in the art and can be introduced into the respective vectors using techniques known to the person skilled in the art (Altschuh et al., Supra).
  • the formation of the mRNAl and the expression of the specific RNA inhibitor are controlled by a regulatable promoter and can be induced by a transcription factor.
  • a regulatable promoter can be induced by a transcription factor.
  • Inducible promoters and the associated transcription factors are known to the person skilled in the art (Harvey D. M. and Caskey C. T .; Curr. Opin. Chem. Biol. 1998 Aug; 2 (4): 512-88)
  • This induction preferably takes place in such a way that the nucleic acid coding for the transcription factor is introduced into the cell and the cell is inhibited under conditions which lead to the expression of the transcription factor.
  • Such conditions are known to the person skilled in the art (Mumberg et al. 1994; Nu leic Acids Res. 22; 5767; Mumberg et al. 1995; Genes 156; 119; Rönicke et al. 1997; Methods Enzymol. 283; 313; Laugwitz et al. 1999; Circulation 99; 925- 933; Schöneberg et al. 1997; J. Clin. Invest. 100 (6): 1547-1556; Hajjar et al. 1997; Circulation Research 81: 145-153).
  • the methods with which the nucleic acid can be introduced into the cell reference is made to the methods listed above for introducing nucleic acids into cells.
  • the nucleic acid coding for the transcription factor is part of a vector.
  • the vector and the particularly preferred vectors, reference is made to the embodiments of vectors mentioned above. The preferred embodiments are as defined above.
  • the vector which contains the nucleic acid coding for the transcription factor can additionally contain a reporter gene, this reporter gene preferably not being the same as the reporter gene which is contained in the other vectors. This makes it possible to evaluate the expression of the transcription factor on the basis of a specific reporter gene signal.
  • the reporter gene reference is made to the above embodiments. The preferred embodiments are as defined above.
  • step a) is carried out before step b) and, following step b), the formation of the mRNAl is induced. Controlled induction of the expression of the nucleic acid construct prevents the cell from being damaged by the toxin before exposure to the potential specific RNA inhibitors.
  • the effectiveness of the specific RNA inhibitor is determined on the basis of the vitality of the cell.
  • “vitality” means the ability of the cell to survive and to grow and proliferate.
  • Methods for determining the vitality are known to the person skilled in the art (Saraste A. and Pulkki K .; Cardiovasc. Res. 2000 Feb; 45 (3): 528-37; Loo D. T, and Rillema JR; Methods Cell. Biol. 1998; 57: 251-64; Witte S .; Dtsch Med. Schuschr. 1967 Sept 29; 92 (39): 1777-81; Darzynkiewicz Z. et al .; Semin. Hematol. 2001 Apr .; 38 (2): 179-93).
  • Optical density is often measured on yeast cells.
  • a cell which contains an effective specific RNA inhibitor shows a higher vitality than cells which do not contain an effective or a less effective RNA inhibitor.
  • a group of cells with an already known, effective RNA inhibitor can also be used as a standard.
  • the proliferation rate of the cells is taken as a measure of the vitality.
  • Methods for measuring the rate of proliferation are known to those skilled in the art (Saraste A. and Pulkki K .; Cardiovasc. Res. 2000 Feb; 45 (3): 528-37; Loo D. T, and Rillema JR; Methods Cell. Biol. 1998; 57: 251-64; Witte S .; Dtsch Med. Schuschr. 1967 Sept 29; 92 (39): 1777-81; Darzynkiewicz Z. et al .; Semin. Hematol. 2001 Apr .; 38 (2): 179- 93).
  • Another object of the present invention is a nucleic acid coding for a toxin and a target protein or a target nucleic acid.
  • This nucleic acid is referred to below as a fusion gene and can be used in particular in the context of the method according to the invention.
  • the fusion gene according to the invention further contains a constitutive or regulable promoter.
  • a constitutive or regulable promoter With regard to the definitions with regard to the promoter, reference is made to the statements in the context of Representation of the method according to the invention referenced. The preferred embodiments are as defined above.
  • the fusion gene according to the invention additionally contains a reporter gene.
  • a reporter gene With regard to the definitions regarding the reporter gene, reference is made to the explanations given in the context of the representation of the method according to the invention. The preferred embodiments are as defined above.
  • the fusion gene according to the invention is part of a vector.
  • the definitions with regard to the vector reference is made to the explanations in the context of the representation of the method according to the invention.
  • the preferred embodiments are as defined above.
  • the fusion gene according to the invention codes for a toxin with a reversible effect.
  • the definitions with regard to the reversible toxin reference is made to the explanations in the context of the representation of the method according to the invention.
  • the preferred embodiments are as defined above.
  • the invention further relates to a cell containing the fusion gene according to the invention.
  • the preferred embodiments of the cell are defined as above in the context of the explanations regarding the method according to the invention.
  • the invention also relates to a test system containing the cell according to the invention.
  • the test system is designed in such a way that it enables parallel testing of a large number of potential specific RNA inhibitors.
  • the cell contained in the test system is a yeast cell.
  • the expression of the toxin can be regulated by an adjustable promoter (see above).
  • an antisense library e.g. shotgun library
  • the test system is preferably present in microtiter plates.
  • the invention further relates to the use of the nucleic acid according to the invention for finding specific RNA inhibitors.
  • the preferred embodiments are as defined above.
  • the invention further relates to the use of a nucleic acid construct containing a nucleic acid coding for a toxin and at least one restriction site suitable for cloning, in particular a multiple cloning site or a recombination cassette, for finding specific RNA inhibitors.
  • a nucleic acid construct containing a nucleic acid coding for a toxin and at least one restriction site suitable for cloning, in particular a multiple cloning site or a recombination cassette, for finding specific RNA inhibitors.
  • a “restriction site suitable for cloning” is to be understood as a restriction site that is located at a suitable site of the nucleic acid construct.
  • a “suitable site” is understood to mean a site in the sequence of the nucleic acid construct, at which the nucleic acid sequence coding for the target protein or the target nucleic acid (for definitions see above) can be introduced in such a way that the expression of the resulting fusion gene is guaranteed.
  • the restriction sites are preferably 5 'from the start codon or 3' from the stop codon of the nucleic acid coding for the toxin.
  • a “recombination cassette” is to be understood as an area which enables homologous recombination with the nucleic acid coding for the target protein and the target nucleic acid (or parts thereof) (for example gateway system; Walhout J. et al., Methods Enzymol , 2000, 328: 575-92).
  • the nucleic acid construct contains a multiple cloning site.
  • cloning sites are known to the person skilled in the art (Altschuh et al., Supra; Molecular Cell Biology, Lodish H. et al .; Fourth Edition W. H. Freeman).
  • RNA inhibitors for the first time, methods and means are provided by the present invention which enable a quick and efficient screening for potential specific RNA inhibitors.
  • the present invention thus makes an important contribution to the development of specific RNA inhibitors and thus to the development of medicaments whose action is based on the suppression of the expression of a protein.
  • Fig. 1 Preferred embodiments of the method according to the invention a) In addition to the use of a fusion gene from a target and a toxin, the same target can also be fused with one or more other toxins. b) Two or more different targets can also be fused with the same or one or more different toxins. c) A fusion gene consisting of several targets and one or more toxins can also be used.
  • Option c) has the advantage that the expression of the toxin is prevented as soon as a suitable RNA-modifying substance becomes effective for one of the targets used; while in embodiment b) it is tested whether effective RNA-modifying substances are also present in the target for all the targets used
  • Fig. 2 Scheme for recombination and amplification of the adenovirus
  • PA polyadenylation signal
  • AV recombinant adenovirus
  • Antisense DNA DNA region complementary to "DCMAG I, 1st binding domain"
  • Fig. 3 Scheme for recombination and amplification of the adenovirus
  • PA polyadenylation signal
  • AV recombinant adenovirus
  • Toxin RNase fusion protein
  • Tatget DNA "DCMAG 1, 1st binding domain”
  • Fig. 4 Scheme for adenoviral infection of cardiac muscle cells
  • AV recombinant adenovirus
  • Fig. 5 Scheme for antisense selection in heart muscle cells
  • An effective antisense RNA prevents expression of the toxic fusion protein by attachment to the m-RNA of the target protein, and the cell can survive. If the antisense construct and mRNA are not duplexed, the toxic fusion protein is formed, which induces cell death.
  • Fig. 6 Analysis of efficient antisense constructs using a fluorescence microscope
  • An effective antisense RNA can be detected in the fluorescence microscope using weakly fluorescent, vital cells. Ineffective antisense constructs are detected using highly fluorescent, dying cells.
  • the recombinant adenovirus was produced according to methods known to the person skilled in the art (Mizugischi, H. et al., Adv. Drug Deliv. Rev. 2001 Nov. 19; 52 (3): 165-76; Danthinne X. and Imperiale M. J .; Gene Ther.
  • cDNAs for TOX gene, sense and antisense constructs Cloning of the sense fragments from cardiac cDNA libraries using suitable primers; likewise the cloning of the antisense fragments; however, these were cloned in the reverse orientation. 5 'or 3' located ribozymes were introduced using primers. The sequences of the antisense were selected “by eye”. When designing the ribozyme, care was taken to ensure that a GUC is located in the target sequences (cleavage site)
  • Neonatal cardiac muscle cells MediGene AG, Martiensried, Germany
  • HEK Human Embryonic Kidney
  • the cDNA against which an antisense construct is to be tested is inserted into two restriction sites of the adenovirus plasmid by means of ligation.
  • An electroporation of 25 ⁇ l ElectroMax DH10B E. coli cells with DNA of the ligation mixture (2.5 kV, 25 ⁇ F, 200 ⁇ , 0.2 cm electroporation cuvette) is then carried out.
  • the cells are incubated shaking in 1 ml of SOC medium for 2 hours at 37 ° C. before the bacterial suspension is plated onto agar plates (ampicillin 100 ⁇ g / ml). After 14 hours, a single colony is picked and, after successful control PCR, a DNA midi preparation of the clone is carried out.
  • AV Plasmid (Pacl digested) are mixed with 240 ⁇ l Optimem. 20 ⁇ l lipofectamine are mixed with 240 ⁇ l Optimem. Both batches are mixed and incubated for 30 minutes at room temperature. The DNA-liposome approach is then placed on a HEK cell culture (25 cm 2 bottle, 50% confluent, in 2.5 ml Optimem). After overnight incubation (37 ° C., 5% CO 2 ), the medium is suctioned off and 5 ml of fresh medium (DMEM, 10% FCS, antibiotic / antifungal) are added. Successful lipofection can be checked by fluorescence microscopy (YFP / CFP emission of the transfected cells).
  • the HEK cells show a cytopathic effect (rounding cells with strong YFP / CFP fluorescence).
  • the cells are pelleted (500x g), suspended in 2 ml of sucrose buffer and the virus released by three subsequent freeze / thaw cycles (N 2 1, 37 ° C. water bath). Cell debris is then pelleted at 500x g and the virus supernatant is stored in cryotubes at - 80 ° C.
  • HEK cell culture 75 cm 2 , 70% confluent. After 4-6d incubation (37 ° C, 5% CO 2 ) the infected cells show a cytopathic effect and the viruses are processed as above (see: Lipofection of adenovirus plasmids). For further amplification, further HEK cell cultures (3 ⁇ 182 cm 2 , 70% confluent) are infected with the adenovirus. After approx. 2 d, the viruses are processed in 2 ml sucrose buffer as described and stored in cryotubes at -80 ° C.
  • an NAP25 column is equilibrated with 25 ml sucrose buffer. 2 ml of virus suspension are added to the column and run into the column bed with 2 ml of buffer. The purified virus is eluted in cryotubes with a further 2 ml of buffer. Storage takes place at -80 ° C.
  • Hearts are isolated from neonatal rats (Wistar rats, l-3d), crushed using a scalpel and trypsinized overnight at 4 ° C. Trypsin digestion is inhibited and the cells are digested with collagenase for 35 min at 37 ° C. The cells are then cleaned on a sieve and, after centrifugation, taken up in a medium containing serum (DMEM / M-199 4/1, horse serum, fetal calf serum, sodium pyruvate, antibiotic, antmycotic). Infection of cardiac muscle cells
  • Neonatal cardiac muscle cells (Wistar rats, l-3d) are sown in a cell density of 25x10 4 cells / cm 2 on polystyrene cell culture dishes. After 24 hours of incubation (37 ° C, 5% CO 2 ), the serum-containing medium is exchanged for serum-free medium (DMEM / M-199 1/4, Na pyravate, antibiotic, antifungal). The cells are then infected with recombinant adenovirus, 4-fold TCJD50 for virus "Antisense” and adenovirus "Tox-Fusion". Successful infection can be detected after> 24 hours using fluorescence microscopy.
  • the infected cardiac muscle cells are examined 48-60 hours after infection using fluorescence microscopy.
  • Cell morphology / cell vitality and fluorescence intensity of the CFP-coupled fusion protein can be analyzed as parameters.
  • a fusion RNA to be inhibited (target RNA + toxin RNA + cyan fluorescent protein RNA) and a complementary antisense RNA are transcribed in cardiac muscle cells.
  • the effectiveness of the antisense nucleotide is determined by reducing the expression of the fusion protein.
  • the parameters of an effective antisense RNA to be analyzed are the increased cell vitality and a reduced fluorescence signal of the cyan fluorescent protein (CFP).
  • the cDNAs to be transcribed in cardiac cells are inserted into two recombinant adenovirus plasmids by means of ligation:
  • - Adenovirus "Antisense” is used for the constitutive expression of the antisense RNA (Fig. 2). Constructs against the N-terminal binding domain of the cDNA "DCMAG I” (MediGene, Dr. Rohrbach) are available as antisense nucleotides.
  • - Adenovirus "Tox-Fusion” is used for the inducible expression of the fusion RNA (target RNA + toxin RNA + cyan fluorescent protein RNA) (Fig. 3).
  • the N-terminal binding domain of the cDNA "DCMAG I" (MediGene) is used as the target gene for the antisense, and an RNase fusion protein from the RNase TIM (from Aspergillus oryzae) and the
  • RNase III (from Escherichia coli) was used.
  • the adenovirus plasmids are amplified and purified in E. coli (midi preparation).
  • the linearized DNA is then packed into recombinant adenoviruses in HEK cells and amplified in further passages.
  • adenovirus "tox fusion" can only be amplified in the non-induced state, since expression of the toxin damages the HEK cells.
  • the two virus constructs are finally column-cleaned and titrated.
  • Neonatal myocardial cells are infected with the two adenoviruses (Fig. 4).
  • the transcription of the fusion protein of the adenovirus "Tox-Fusion" is achieved by induction of the regulatable promoter. After approximately 24 hours, the infection of the cells is checked on the fluorescence microscope (CFP signal for expression of the fusion protein).
  • the constitutively transcribed antisense RNA can now bind to the target area of the fusion RNA to form a double strand (Fig. 5, right).
  • An efficient antisense RNA prevents expression of the fusion protein, so that no or very little toxin is produced in the cell.
  • the CFP fluorescence signal in the cell decreases.
  • An ineffective antisense RNA cannot inhibit the expression of the fusion protein, the cells initially show an increased CFP signal and then die due to the effects of toxins (Fig. 5, left).
  • the screening for effective antisense RNAs can thus be easily carried out using the fluorescence roscopically or with the help of a laser scanning xytometer (LSC) (Fig. 6).
  • the antisense constructs identified as effective in the screening can finally be modified or optimized and analyzed in further test cycles.
  • the system can also be used in all other cells that can be infected by adenovirus.
  • the selection system can therefore be used in high-throughput screening in microtiter plates (e.g. HeLa cells) or in vivo (animal models).

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Abstract

L'invention concerne un procédé permettant d'identifier des substances, adaptées pour inhiber spécifiquement l'expression d'une protéine ou l'action d'un acide nucléique. L'invention concerne en outre un ARN codant une toxine et une protéine cible ou un acide nucléique cible, ainsi que son utilisation pour identifier des inhibiteurs d'ARN spécifiques.
PCT/EP2003/002350 2002-03-08 2003-03-07 Procede d'identification d'inhibiteurs d'arn specifiques WO2003076668A2 (fr)

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Publication number Priority date Publication date Assignee Title
WO2022200407A1 (fr) * 2021-03-25 2022-09-29 Martin-Luther-Universität Halle-Wittenberg Identification fiable de zones (sites-a) dans des molécules d'arn complexes qui sont accessibles à des acides nucléiques ou des complexes d'acides nucléiques avec endonucléases

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EP0436467A3 (en) * 1989-12-29 1992-10-21 Ciba-Geigy Ag Expression of s-locus specific glycoprotein gene in transgenic plants
AUPM516994A0 (en) * 1994-04-20 1994-05-12 Gene Shears Pty. Limited An in vivo gene expression system
US5733726A (en) * 1995-06-07 1998-03-31 Emory University Cytotoxicity-based genetic selection system (TOXSEL)
DE19905501B4 (de) * 1999-02-10 2005-05-19 MediGene AG, Gesellschaft für molekularbiologische Kardiologie und Onkologie Verfahren zur Herstellung eines rekombinanten Adeno-assoziierten Virus, geeignete Mittel hierzu sowie Verwendung zur Herstellung eines Arzneimittels
US20020002278A1 (en) * 1999-11-05 2002-01-03 Timothy W. Nilsen Random intracellular method for obtaining optimally active nucleic acid molecules
US20010031485A1 (en) * 2000-03-22 2001-10-18 Sibtech, Inc. Recombinant proteins containing Shiga-like toxin and vascular endothelial growth factor fragments

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022200407A1 (fr) * 2021-03-25 2022-09-29 Martin-Luther-Universität Halle-Wittenberg Identification fiable de zones (sites-a) dans des molécules d'arn complexes qui sont accessibles à des acides nucléiques ou des complexes d'acides nucléiques avec endonucléases

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