WO2002024931A1 - Systeme d'expression pour acides nucleiques fonctionnels - Google Patents

Systeme d'expression pour acides nucleiques fonctionnels Download PDF

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WO2002024931A1
WO2002024931A1 PCT/EP2001/010905 EP0110905W WO0224931A1 WO 2002024931 A1 WO2002024931 A1 WO 2002024931A1 EP 0110905 W EP0110905 W EP 0110905W WO 0224931 A1 WO0224931 A1 WO 0224931A1
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nucleic acid
acid sequence
rna
box
cell
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PCT/EP2001/010905
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German (de)
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Michael Blind
Michael Famulok
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Nascacell Gmbh
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a nucleic acid sequence for expressing a nucleic acid to be inserted therein, an expression system comprising this nucleic acid sequence, a vector comprising this, a cell comprising this and a transgenic animal and transgenic plant comprising this.
  • the invention further relates to various uses of the nucleic acid sequence according to the invention.
  • RNA molecules have recently been used to modulate intracellular processes. For example, the translation of proteins can be blocked by antisense RNA which hybridizes with the coding RNA (review article: Mesmaeker et al., Acc. Chem. Res. 28 (1995), 366-374).
  • a comparable mechanism works catalytically active ribozymes such as hammerhead or hairpin ribozymes, which can attach to complementary regions on mRNAs and destroy their target RNA by hydrolysis of phosphodiester bonds (Castanotto et al., Advances Pharmacol. 25 (1994), 289-317 ; Rossi, Tibtech 13 (1997), 301-305).
  • RNA molecules particularly interesting for applications in gene therapy, which opens up a broader field of application than simply replacing missing or mutated genes for proteins.
  • Antisense nucleic acids and ribozymes do not work in gene therapy by expressing an essential, missing protein, but act directly on molecules expressed in the target cell.
  • the RNA molecules are mostly used to inhibit the translation of proteins which, due to unnatural expression, cause a certain lack of awareness or are required by pathogens, such as viruses, for their life cycle.
  • a 900 nucleotide long antisense RNA was tested against the CaSm oncogene occurring in pancreatic cancer (Kelley et al., Surgery 128 (2000), 353-60). Recombinant adenoviruses were used as vectors.
  • the in vitro proliferation of PC cell lines was reduced by up to 93% through the antisense constructs.
  • tumor growth in mouse models could be reduced by 40% by mtratumoral injection and the average survival rate increased from 35 to 60 days.
  • Anti-t ⁇ t or double anti-t t and -rev hammerhead ribozymes for anti-HIV therapy were also tested in a clinical study. Due to the degradation of the Tat and Rev RNA in the CD 4+ T cells that were transgenic as a result of the treatment, the corresponding proteins were not formed and the replication of the viruses could not take place. Such transgenic CD4 + T cells can survive infection with HIV. In the clinical experiments, CD4 + T cells from patients were transduced with the ribozymes and returned to the patients. The cells modified in this way could still be found in the patients after 10 months (Amado et al, Front. Biosci. 15 (1999), D468-475).
  • ribozymes which were directed against the RNA component of telomerase or the Bcr-Abl oncogene, were also used in therapeutic approaches to combat cancer cells (Yokoyama et al., Cancer res. 58 (1998), 5406-5410; Snyder et al., Blood 82 (1993), 600-605; Wright et al, Antisense Nucleic Acid Drag Dev 8 (1998), 15-23).
  • RNA molecules Another promising class of functional RNA molecules are nucleic acid ligands, in particular RNA ligands, which can bind specifically to intracellular proteins and directly inhibit their activity.
  • nucleic acid ligands can either be naturally occurring RNA motifs which, by overexpression, compete for the binding of an endogenous cellular nucleic acid, in particular RNA, to their interaction partner, or new aptamers generated by in vitro selection, with aptamers on both RNA and DNA. Basis exist.
  • aptamers could be isolated against a number of proteins, whether naturally nucleic acid-binding or not, by the in vitro selection process (or also SELEX for "systematic evolution of ligands by exponential enrichment") (review article: Gold et al., Annu Rev. Biochem. 64 (1995), 763-797; Ellington and Conrad, Biotechnol. Annu. Rev. 1 (1995), 185-214; Famulok and Mayer, Curr. Top. Microbiol. Immunol. 243 (1999), 123-36).
  • both a 27 nucleotide long sequence of the natural interaction domain of the viral Tar RNA element and a de novo isolated anti-Tat aptamer were used to inhibit the Tat transactivator protein of HIN (Bohjanen, Nucleic Acid Res. 24 (1996), 3733 -3738; Yamamoto et al., Genes Cells 5 (2000), 371-88).
  • the binding of the Tat protein to the natural TAR-RNA element both TAR-1 and TAR-2 was competed specifically and the Tat-dependent transactivation in vitro and in vivo was strongly inhibited.
  • aptamers were first examined in a transgenic animal model.
  • the aptamers directed against the protein B52 involved in the splicing were able to induce the same phenotype in the fruit fly as the corresponding genetic knock-out and to compensate for the effects of the overexpression of the B52 protein (Shi et al., Proc. latl. Acad. Sci., 96 (1999), 10033-10038).
  • aptamers against the cytoplasmic domain of the CD 18 integrin subunit in human leukocytes to inhibit a signal transduction protein which did not naturally interact with nucleic acids and thus to interrupt the signal cascade in the leukocytes which activated the CD 18 integrin receptors and the cells adhered to them natural ligand ICAM-1 ("intracellular adhesion molecule-1") (Blind et al., Proc Natl. Acad. Sei. USA 96 (1999), 3606-3610).
  • aptamers are discussed as effective modulators of the function of proteins, which can be used to study the cellular proteome or in therapeutic approaches.
  • the latest results show that aptamers can also be used with great success especially against intracellular target molecules.
  • These modulators also called intramers (intracellular aptamers)
  • intramers intracellular aptamers
  • Expression systems are used that ensure an effective transcription of the molecules in a eukaryotic cell background.
  • RNA polymerase-I for example, transcribes the genes of the large ribosomal rRNA subunits, RNA polymerase-II mRNAs and RNA polymerase-III the genes of various small ribonucleic acids
  • RNA polymerase II and III promoters from eukaryotic and viral systems or purely viral transcription units such as bacteriophage or poxvirus promoters were used (Cheetham GM, et al., Curr Opin Struct Biol. 10 (2000), 117 -123; Chakrabarti S, et al, Biotechniques. 23 (1997), 1094-1097).
  • RNA polymerase I promoters do not appear to be suitable for the expression of functional nucleic acids, since the transcription units are relatively complex. Additional protein factors that bind to multiple terminator sequences are required in particular for the termination of the transcripts. In addition, the transcripts are partially processed by nucleases during termination. (Paule and White, Nucleic Acid Res. 28 (2000), 1283-1298). It therefore seems difficult to develop reliable systems that allow a defined termination of the transcripts. In addition, the transcription of the RNA polymerase I transcription units takes place in specialized compartments, the nucleoli.
  • ribosomal RNAs are further processed by nucleases and RNA editing complexes and assemble into large ribosomal complexes (Olson et al., Trends Cell Biol. 10 (2000), 189-196). However, these modifications could adversely affect the integrity of other nucleic acid sequences to be expressed.
  • Pol II promoters can be seen in the very different levels of transcription efficiency of the individual promoters and the widespread cell type specificity, ie the dependence on transcription factors which are only expressed in certain cell types.
  • the long transcripts are usually strong processed and associated with many multifactorial complexes, such as the splicing machinery or the ribosomes.
  • the largely intactness of the primary transcript and the largely freedom from interfering associations with cellular factors are desirable for the maintenance of the actual activity, eg binding of the specific cellular target molecule.
  • RNA polymerase III promoters seem to be more suitable for the expression of functional RNAs. They are responsible for the transcription of many small RNA molecules in eukaryotic cells and are active in all cell types. In addition, they can be found ubiquitously in all eukaryotic species in homologous RNA transcription units. Pol III promoters control the transcription of small RNA such as tRNA, snRNA, snoRNA, 5S rRNA, or small viral RNA, such as adenoviral NA RNA. Another advantage of the RNA-Pol LU promoters is their extremely high transcription activities. Natural transcripts accumulate in cells up to 10 5 to 10 6 molecules per cell.
  • RNA molecules For example, expression levels of 400,000 molecules per cell are achieved by the U6 snRNA (Weinberg and Peuman, J. Mol. Biol. 38 (1968): 289-304). Therefore, Pol III transcription units were used for the expression of foreign RNA molecules as well (review article: Couture and Stinchcomb, TIG Y2 (1996), 510-514; Rossi, Tibtech 13 (1995), 301-305; Braage et al , Tibtech 16: 434-438 (1998).
  • An ideal expression cassette for functional RNA molecules should combine several points: i) high cellular expression levels, ii) preservation of the functional properties of the expressed RNA molecule, iii) colocalization with the cellular target molecule.
  • nucleic acid ligands against the viral Rev protein and the cytoplasmic domain of the CD18 integrin subunit have already been carried out compact, flanking stem loop sequences stabilized (Good et al., Gene Ther. 4 (1997), 45-54; Blind et al, Proc. Natl. Acad. Sci. USA 96 (1999), 3606-3610).
  • RNA sequences such as tRNA, U6-RNA, retroviral or mRNA constructs, were also used to transport the ribozyme RNA to certain compartments.
  • RNA species such as mRNA, tRNA, small viral RNAs or ribosomal RNA
  • RNA species such as mRNA, tRNA, small viral RNAs or ribosomal RNA
  • the cytoplasmic or nuclear localization is believed to depend on two balanced mechanisms, core retention and active transport into the cytoplasm (Schmidt-Zachmann et al., Cell. 74 (1993), 493-504; Custodio et al, EMBO J. 18 (1999), 2855-2866).
  • various mechanisms are responsible for exporting different RNA classes (rRNA, mRNA, snRNA and tRNA) from the nucleus into the cytoplasm (Jarmolowski et al., J. Cell. Biol.
  • tRNAs form complexes with the export factors Xpo-t and the small G protein Ran in its GTP-bound form (Ran-GTP) (Hutay et al., Mol. Cell. 1_ (1998), 359-369, Mol. Cell. Biol. 18 (1998), 6374-6386) and are transported through the nuclear pores into the cytoplasm.
  • the present invention has for its object to provide an expression system that allows the expression of functional nucleic acids. Another object is to provide an expression system that allows compartment-specific expression of functional nucleic acids. Finally, it is an object of the invention to provide an expression system which allows the expression of functional nucleic acids in all desired tissues.
  • the object is to provide an expression system which is active in a host cell without the host cell having to be provided with further proteins which are necessary for the expression, in particular transcription, of the functional nucleic acid.
  • nucleic acid sequence for expressing a nucleic acid to be inserted into the nucleic acid sequence, the nucleic acid sequence comprising the following elements in the 5 '- 3' direction:
  • the Cl motif and the C2 motif together form a helix;
  • the AI box comprises k bases, k independent of 1 and m being an integer from 0 to 100;
  • the A2 box comprises 1 bases, where 1 is an integer from 0 to 100 regardless of k and m; and
  • the A3 box comprises m bases, where m is an integer from 0 to 20 regardless of k and 1.
  • k is an integer from 0 to 20, preferably from 5 to 15, independently of 1 and m;
  • 1 is an integer from 0 to 20, preferably from 5 to 15, independently of k and m; and m is an integer from 0 to 9 regardless of k and 1.
  • the Cl motif comprises n bases, where n is an integer> 10 independently of p, k, 1 and m and the C2 motif comprises p bases, where p is an integer> 10 independently of n, k, 1 and m is.
  • n is an integer> 16, preferably> 20 regardless of p, k, 1 and m and p is an integer> 16, preferably> 20 regardless of n, k, 1 and m.
  • the double helix formed by Cl and C2 together comprises at least 10 base pairs.
  • the terminator is a terminator for RNA polymerase III.
  • the terminator comprises four or more successive uridine bases in the event that the nucleic acid sequence is an RNA sequence and four or more successive thymidine bases in the case that the nucleic acid sequence is a DNA Sequence is.
  • the nucleic acid sequence according to the invention can further comprise a promoter, in particular a promoter for RNA polymerase III.
  • the promoter is selected from the group comprising promoters of 5S-RNA genes, U6 sn RNA promoters, tRNA promoters, 7 SL-RNA promoters and VA-RNA promoters ,
  • the nucleic acid sequence further comprises the nucleic acid to be inserted, the nucleic acid to be inserted preferably being arranged between the AI box and the A2 box.
  • the nucleic acid to be inserted is a functional nucleic acid.
  • the functional nucleic acid is selected from the group comprising aptamers, intramers, aptamzymes, allosteric centers of aptazymes and ribozymes.
  • the functional nucleic acid interacts with a target molecule, in particular a nucleic acid target molecule, via a mechanism that is different from complementary base pairing.
  • the object is achieved by an expression system according to the invention, which comprises a nucleic acid sequence according to the invention.
  • the expression system is a functional nucleic acid for the expression, preferably for the transcription.
  • the task is solved by an expression system for the
  • the AI box comprises k bases, k independently of 1 and m being an integer from 0 to
  • the A2 box comprises 1 bases, where 1 is an integer from 0 to regardless of k and m
  • the A3 box includes m bases, where m is an integer from 0 to independent of k and 1
  • the Cl motif comprises n bases, where n is an integer> 10 regardless of p, k, 1 and m and the C2 motif comprises p bases, where p is an integer> 10 regardless of n, k, 1 and m is.
  • the object is achieved by a vector comprising a nucleic acid sequence according to the invention or an expression system according to the invention.
  • the object is achieved by a cell comprising a nucleic acid sequence according to the invention or an expression system according to the invention or a vector according to the invention.
  • the cell is a eukaryotic cell.
  • the eukaryotic cell is preferably Saccharomyces cerevisiae. In another preferred.
  • the cell is an oocyte from Xenopus laevis. In a further preferred embodiment, the eukaryotic cell is a mammalian cell.
  • the cell is a human cell.
  • the cell is a plant cell.
  • the object is achieved by a transgenic animal comprising a cell according to the invention.
  • the transgenic animal is selected from the group comprising mammals, fish, insects and nematodes.
  • the transgenic animal is a mammal.
  • the mammal is preferably selected from the group comprising mice, rats, rabbits, dogs, pigs, sheep, cows, horses, goats, donkeys, camels, chickens and monkeys.
  • the animal is a nematode, preferably C. elegans.
  • the animal is a fish, preferably a zebra fish.
  • the animal is an insect, preferably Drosophila melanogaster.
  • the animal is a frog, in particular Xenopus laevis or early multicellular stages thereof.
  • the object is achieved by a transgenic plant comprising a cell according to the invention.
  • the plant is selected from the group consisting of useful plants, vegetables, rice, wheat, corn, manioc, potatoes, millet, soy, tomatoes, cotton, peas, tobacco, beans and Aradopsis fhaliana.
  • the object is achieved by using the nucleic acid according to the invention for target validation
  • the object is achieved by using the nucleic acid according to the invention for target identification
  • the object is achieved by using the nucleic acid according to the invention for gene therapy.
  • the nucleic acid sequence according to the invention is the expression system according to the invention.
  • the object is achieved by a method for gene therapy treatment of an organism, wherein the organism is administered a nucleic acid sequence according to the invention or an expression system according to the invention or a vector according to the invention or a cell according to the invention.
  • a medicament comprising a nucleic acid sequence according to the invention and / or an expression system according to the invention and / or a vector and / or a cell according to the invention.
  • nucleic acid according to the invention allows efficient expression of functional nucleic acids, i.e. a high level of expression, functional or functional contracts and targeted localization.
  • motif refers here to a sequence of nucleotides which are covalently linked to one another, typically via a phosphodiester bond
  • the term “motif” is also preferably used herein in the sense that it denotes a polynucleotide comprising at least two nucleotides covalently linked to one another, the polynucleotide either as such or if it is associated with a other chemical compound such as another motif, ie another polynucleotide, interacts with a secondary or tertiary structure.
  • the Cl motif and the C2 motif interact with each other and form an overall helix.
  • sequence of the nucleotides building them up is generally determined by the interaction partner and / or the secondary and / or tertiary structure to be trained.
  • one strand can consist of a sequence of cytosine residues and the second strand required for forming the helix can consist of a sequence of guanosine residues.
  • the selection of a specific sequence for a motif is therefore within the capabilities of the experts.
  • box as used in particular herein also denotes a sequence of nucleotides which are covalently linked to one another, typically via a phosphodiester bond, and are therefore present as a polynucleotide.
  • This polynucleotide has in a nucleic acid in which it contains is, as a rule, the function of separating certain other parts of the nucleic acid, such as the motifs, from one another, arranging them relative to one another, connecting them to one another or arranging them in such a way that the required or desired secondary and / or tertiary structure is formed
  • the sequence of the boxes can be determined by the experts in accordance with certain selection criteria depending on the specific task that the box has to perform in the respective case, that is to say depending on the context in which it is to be built in.
  • the box itself does not interact with the other parts of the nucleic acid according to the invention which can be used as an expression system and does not block, for example, does not block the formation of the secondary and / or tertiary structures required for the functionality of the nucleic acid according to the invention hybridizes the Cl motif, which is to hybridize with the C2 motif to form a helix.
  • the invention is based on the surprising finding that the nucleic acid sequence or nucleic acid according to the invention or the expression system comprising this, which is also referred to below as the Pol-ffl helix expression system, is the reliable expression of nucleic acids or of nucleic acid ligands which are incorporated in the Expression system can be inserted or inserted, allowed in eukaryotic cells.
  • the invention comprises a structural motif which consists of a terminally arranged helix which is formed by base pairing of the 5 'end and the 3' end of the expression construct.
  • the functional nucleic acid to be expressed is enclosed by this terminally arranged helix.
  • the expression system according to the invention and thus also the nucleic acid sequence according to the invention combines in a unique way all properties which are required for an effective expression of the functional nucleic acid molecules, typically an RNA molecule.
  • the following properties characterize the inventive Pol III helix expression system: i) the structure of the inserted functional nucleic acid [RNA] and thus its ability to interact three-dimensionally with its binding partners is maintained, ii) the rigid structure of the terminal helix protects the expression contract from the Degradation by intracellular nucleases, iii) by small variations of the stnjk. ⁇ u__dßr_ form that interact with structures, preferably complementary structures, on other molecules.
  • the nucleic acid ligands can be DNA nucleic acid sequences, RNA nucleic acid sequences or chemically modified forms of DNA or RNA nucleic acid sequences.
  • nucleic acids can also include “foreign” nucleic acids, that is to say those nucleic acids which, owing to their defined secondary and tertiary structures, form three-dimensional contact areas which interact with structures, preferably complementary structures, on other molecules, and as such in the cellular background do not occur naturally.
  • the term functional nucleic acids can also include those nucleic acids that, due to their defined secondary and tertiary structures, form three-dimensional contact surfaces that interact with structures, preferably complementary, three-dimensional structures, on other molecules, and as such already occur in the cellular background, whereby that Occurrence can be traced back to the fact that the respective cell was already transformed at an earlier point in time, that the respective cell already carries such a functional nucleic acid as part of its natural genetic equipment, or that the respective cell has such a functional nucleic acid as a result of a pathological event to which the cell is or has been subjected.
  • the term “functional nucleic acids” can also include those preferably foreign nucleic acid molecules, in particular RNA molecules, which are introduced into cells and which then influence their function through interaction with cellular factors. Functional in this context, alternatively or cumulatively to what has been said above, can also mean that the nucleic acid molecules are able to influence cellular components such as other nucleic acids (such as deoxyribonucleic acids or ribonucleic acids) or proteins by binding or catalytic activity. These functional nucleic acids can be used, for example, for therapeutic, diagnostic applications, the validation of the task of intracellular components, for the identification of functionally important cellular factors, for the inhibition of certain proteins in transgenic organisms for the purpose of increased productivity or other purposes. Examples of functional ribonucleic acids include antisense molecules, ribozymes, aptamers or fritramers.
  • Nucleic acid ligands are also preferably to be understood here as meaning single-stranded nucleic acids which form three-dimensional motifs through defined secondary and tertiary structures and which come into contact specifically with other molecules, in particular those which have a structurally complementary surface, via hydrogen bonds, electrostatic and hydrophobic interactions or others , These interactions across three-dimensional motifs were confirmed by a large number of elucidated structures (review article: Famulok, Curr. Opin. Struct. Biol. 9 (1999), 324-329; Nagai and Mattaj (Eds.), RNA protein interactions (1994) Oxford University Press; The RNA World Website at IMB Jena, www.imb-jena.de/RNA).
  • Nucleic acid ligands can be naturally occurring motifs, that is to say naturally occurring nucleic acid sequences.
  • the RRE rev responsive element
  • HIN human immundeficiency virus
  • the viral Rev protein interacts with the viral Rev protein and thus mediates the export of R ⁇ A from the cell nucleus into the cytoplasm (Malim et al, ⁇ ature 338 ( 1989), 254-257; Cochrane et al., Proc. Latl. Acad. Sci. USA 87 (1990), 1198-1202).
  • IRE motif iron responsive element
  • non-naturally occurring nucleic acid ligands are in vitro selected aptamers or the allosteric centers of aptazyme (hybrid molecules from aptamers and ribozymes, for example described by Robertson MP, et al., Nucleic Acids Res. 28 (2000), 1751-1759), which by Binding of a ligand regulate the catalytic activity of the aptazyme.
  • Nucleic acid ligands in the sense of the present invention are in particular also those nucleic acids which bind to a target molecule by means of a mechanism or which recognize the target molecule by means of a mechanism which is different from the mechanism of complementary base pairing. This also applies if the target molecule is a Is nucleic acid.
  • target is to be understood here as a molecule which interacts with the nucleic acid ligand.
  • nucleic acid ligand The various definitions or aspects of the term nucleic acid ligand given above can be used herein both individually and in any combination.
  • Aptamers are to be understood here to mean in particular single-stranded, high-affinity nucleic acid ligands which are derived from the technology of in vitro selection or SELEX ("systematic evolution of ligands by exponential enrichment") developed in the early 1990s (Tuerk and Gold, Science 249 (1990) , 505-510) are isolated.
  • SELEX systematic evolution of ligands by exponential enrichment
  • aptamers which consist of ssDNA or ssRNA, have very high affinities and specificities for their target molecules.
  • numerous of these functional nucleic acids have been isolated for a large number of small, organic compounds, peptides, proteins, or complex structures such as viruses and cells (review article: Gold et al., Annu. Rev. Biochem.
  • the high potential of the technology lies in the in vitro process of aptamer selection.
  • the nucleic acid ligands are enriched from combinatorial libraries of up to 10 15 individual sequences by repeated cycles of contact with the target molecule, separation of all non-binding nucleic acids and enzymatic amplification of the molecules interacting with the target molecule (review article: Klug and Famulok, Mol. Biol. Rep. 20 (1994), 97-107; Conrad et al., Mol. Diversity 1 (1995), 69-78).
  • RNA aptamers from libraries of completely or partially randomized sequences takes place, for example, using affinity chromatography.
  • Other commonly used separation techniques for RNA / protein complexes are electrophoretic separation processes or retention on nitrocellulose membranes.
  • aptamers selected for proteins are able to inhibit their biological activity.
  • a number of nucleic acid ligands for hormones and growth factors such as bFGF ("basic fibroblast growth factor”), hTSH ("human thyroid stimulating hormone”) or vasopressin have been isolated, which bind them to their natural Block receptors and their biological activity (Jellinek et al., Proc. Nati. Acad. Sci. USA 90 (1993), 11227-11231; Lin et al, Nucleic Acid Res. 24 (1996), 3407-3413; Williams et al ., Proc. Nati. Acad. Sci. USA 94 (1997), 11285-11290).
  • Intramers are to be understood here as meaning those aptamers which, in addition to the pure binding of their ligand, have been specifically designed for use in the intracellular environment.
  • aptamers can be stabilized, transcribed with high transcription efficiency and localized in the cytoplasm by viral polymerases and their target molecules there bind (Blind et al., Proc. Nati. Acad. Sci. USA 96 (1999), 3606-3610).
  • intramellers are to be understood as meaning in vitro selected aptamers which are optimized and used by stabilization, localization, expression, addition of natural or unnatural nucleic acid sequences, chemical modifications or other measures for the purpose of functional modulation of the activity of an intracellular target molecule.
  • Aptazymes are to be understood here in particular as meaning catalytic nucleic acids, so-called ribozymes, which consist of a catalytic domain and an allosteric domain which controls the catalytic activity of the aptazyme by binding an effector molecule.
  • the mechanism is comparable to the regulation of allosterically regulated protein enzymes.
  • Ribozymes can accelerate a number of chemical reactions, mostly phosphoester transfer reactions (Scott, Curr. Opin. Struct. Biol. 8 (1998), 720-726; Carola and Eckstein, Curr. Opin. Chem. Biol. 3 (1999), 274 -283).
  • a series of aptazymes could be produced by design or in vitro selection, the catalytic activity of which is either activated or inhibited by binding a ligand to an additional nucleic acid domain (Soukup and Breaker, Curr. Opin. Struct. Biol. 10 (2000), 318- 325).
  • Aptazymes could be isolated by in vitro selection which contain a new binding domain for ligands which was not previously known (Robertson and Ellington, Nat. Biotechnol. 17 (1999), 62-66; Piganeau et al., RNA 2000, The Annual Meeting of the RNA Society (2000), Madison, (Abstract)). This binding domain can be isolated and used like an aptamer to modulate the corresponding target molecule.
  • Vectors are to be understood here generally as gene transfer systems which are capable of introducing nucleic acids into a host organism such as a cell, preferably a eukaryotic cell, and allow the introduced nucleic acid to be present in the cell in a stable manner and, if appropriate, stable for at least a certain time is expressed.
  • a host organism such as a cell, preferably a eukaryotic cell
  • the various common vector systems are known to those skilled in the art and are described, for example, in Sambrook et al. Molecular Cloning: A Laboratory Manual 3 (1989), Edition: 02, Cold Spring Harbor; Glover, D, DNA cloning: a practical approach: expression Systems (1995), Edition: 02, rl Press.
  • the nucleic acid sequence according to the invention has a number of elements which are optionally present, such as the AI box, the A2 box or the A3 box.
  • the presence or absence of one of these boxes is independent of the presence or absence of one of the other boxes.
  • the A2 box is also preferably present and vice versa.
  • the size or length of the AI box or the A2 box and thus the value of k and 1, each of which describes the length of the AI box or the A2 box, can be different. However, it is preferred if k and 1 have the same value.
  • k and 1 can take any integer value from 0 to 100. Particularly preferred ranges for k and 1 are, independently of one another, 0 to 20, the range from 5 to 15 being particularly preferred.
  • the length of the A3 box expressed as the number of bases m forming the box, can have a value from 0 to 20. A range from 0 to 9 is particularly preferred.
  • the length m of the A3 box can therefore take any integer value between 0 and 20. In principle, a longer length also appears possible, based on the currently available results, although this is not intended to be a limitation in terms of length, there is an upper limit between 10 and 17 if the transcripts are to be exported from the core becomes. If localization in the cell nucleus is desired, the length of box A3 can be greater than 10, the length of box A3 is preferably greater than 15 and more preferably greater than 18. Under these conditions, values of 40, 60 or up to 100 bases are possible in principle.
  • the de facto upper limit for the length of A3 arises when the length of the A3 box changes the structure of the nucleic acid sequence according to the invention or of the expression system according to the invention and in particular the secondary structure shown in FIG. 2A no longer develops.
  • This aspect of the upper limit of m also applies mutatis mutandis to all other running parameters such as k, 1, m, n and p that indicate the length of motifs or boxes, whereby this criterion also applies to the lower limit of said running parameters.
  • the size of the Cl motif as well as the size of the C2 motif, also expressed as the number of bases n or p forming the respective motif, can be configured independently of one another.
  • the value of n or p can each be greater than or equal to 10.
  • a length greater than or equal to 16 is preferred, with a length greater than or equal to 20 being particularly preferred.
  • the length of the Cl motif is equal to the length of the C2 motif.
  • the sequence of the Cl motif and the C2 motif is to be designed in such a way that there is an area within each C motif that is complementary to an area of the other C motif. It is within the scope of the present invention that the mutually complementary regions of the C motifs within the respective C motif are arranged relative to one another at corresponding locations.
  • the mutually complementary regions of the C motifs are arranged at different locations within the respective C motif. This would have the consequence, for example, that the overhangs of the two C motifs - based on the trained helix - could be different.
  • a double helix is formed. It is particularly preferred if the double helix comprises at least 14 base pairs.
  • Base pairings are to be understood here as meaning both Watson-Crick base pairings and non-Watson-Crick base pair pairs, the number of base pairings being able to be composed of any combination of the two base pairing types mentioned above and, in an extreme case, only Watson-Crick base pairings and in another extreme case, only non-Watson-Crick base pairings form the double helix.
  • the actual sequence plays a less important role in the above-mentioned elements, ie the various C motifs and A boxes, of the nucleic acid sequence according to the invention. Rather, the said elements are essentially important because of their structural properties, which leads to the formation of a nucleic acid sequence environment for the nucleic acid ligand to be expressed.
  • sequences which are not complementary to the A boxes or the insert nucleic acid, in particular insert RNA are particularly preferred, so that no stable helices are formed there.
  • Cl and C2 motifs which do not form a perfect helix are preferred, since it is known in the prior art that some intracellular enzymes can be activated by double-stranded RNA.
  • the activation of the dsRNA activated protein kinase (PKR) leads, for example to a blockade of translation, that is, the formation of new proteins and can induce apoptosis (Kaufman RJ, Proc Nati Acad Sei US A. 96 (1999), 11693-11695; Williams BR, Oncogene.
  • dsRNA regions in the helix formed by the Cl and C2 motifs should preferably be no longer than 12 positions, more preferably no longer than 8 positions, the total length of the helix still being 10 Base pairs, and preferably 14 base pairs. This can be achieved, for example, by two differently long Cl and C2 motifs. In this case, some bases of the longer C motif remain unpaired, which leads to an interruption of the continuous helix, for example in Figure 9c in the Pol III helix expression construct PH7, the unpaired base regions G and UU.
  • Another possibility for interrupting a continuous helix is in areas in which non-complementary bases face each other.
  • an unpaired region is formed, which depending on its length is also referred to as a mismatch or internal loop and is flanked by continuous areas of the helix, such as the unpaired bases A and G in the helix of the contracts in Figures 8 a) b) c) or 9c) or 10.
  • the terminators to be used are typically RNA polymerase III terminators. Such terminators are described, for example, in Paule and White, Nucleic Acid Res. 28 (2000), 1283-1298.
  • any terminator is suitable which ensures that the expression and in particular the transcription of the expression system or of the expression system therein inserted and to be expressed nucleic acid, preferably the nucleic acid coding for the nucleic acid ligand, takes place.
  • the terminator can thus also be such a terminator which is specific for another polymerase system, but which is also effective in the expression system according to the invention in the sense that the expression on the terminator or in its vicinity is ended.
  • terminators in which the termination is intrinsically terminated by short DNA sequences are also suitable, using the respectively specific polymerase promoters.
  • sequence-dependent terminators can be found, for example, in the bacteriophage T7 RNA polymerase (Hartvig and Christiansen, EMBO J. 15 (1996), 4767-4774).
  • the nucleic acid sequence according to the invention or the expression system according to the invention comprising this may also comprise a promoter, the promoter preferably being one which controls the expression and particularly the transcription of the nucleic acid ligand inserted in the expression system and thus to be expressed (or the nucleic acid coding for it) , Basically, all such promoters can be used.
  • RNA polymerase III promoters are described, for example, in Paule and White, Nucleic Acid Res. 28 (2000), 1283-1298 and are known to those skilled in the art.
  • Such vectors are particularly suitable if they are compatible with the structural and functional requirements of the Pol III helix expression system according to the invention disclosed herein.
  • RNA polymerase III promoters are the promoters of 5S RNA genes (type 1) (Specht et al., Nucleic Acid Res. 19 (1991), 2189-2191), tRNA promoters (type 2) (Thompson et al., Nucleic Acid Res. 23 (1995), 2259-2268; Sullenger et al., J. Virol. 65 (1991), 6811-6816) U6 snRNA (type 3) (Das et al, EMBO J. 7 (1988), 503-512, Lobo and Hernandez, Genes Dev.
  • type 1 Specific et al., Nucleic Acid Res. 19 (1991), 2189-2191
  • tRNA promoters type 2 (Thompson et al., Nucleic Acid Res. 23 (1995), 2259-2268; Sullenger et al., J. Virol. 65 (1991), 6811-6816)
  • U6 snRNA type 3 (Das et al, EMBO J.
  • RNA molecules have been used for the expression of RNA molecules (review article: Rossi, Tibtech 13 (1997), 301-305; Bramlage et al., Tibtech 16 ( 1998), 434-438).
  • nucleic acid sequence according to the invention or the expression system according to the invention can be provided with a further element which controls the site-specific localization of the nucleic acid ligand.
  • Such localization-controlling elements are within the nucleic acid sequence according to the invention or the expression system can be arranged in the A-boxes (1-3).
  • Such elements are known to those skilled in the art and are described, for example, in Cullen, Mol Cell Biol. 20 (2000), 4181-4187 or in Pederson, FASEB J. 13 Suppl 2 (1999), 238-242.
  • the RRE rev responsive element
  • the viral Rev protein which is involved in the transport of the viral RNAs from the cell nucleus into the cytoplasm
  • tRNAs complexes with the export factors Xpo-t and the small one G-protein Ran in its GTP-bound form (Ran-GTP) (Hutay et al., Mol. Cell. J_ (1998), 359-369, Mol. Cell. Biol.
  • nucleic acid sequence according to the invention which could also be referred to as nucleic acid
  • sequence of the individual is important Sections of the nucleic acid sequence or the expression system to the extent that, as stated above, they can be of importance for the localization signal or the sequence can comprise an intragenic promoter or promoter element.
  • the sequence is also of importance in so far as it has an effect on the formation of the required secondary structure, in particular in interaction with other elements and the sequences building them up.
  • the selection of a suitable sequence with the aim of generating the secondary structure can be determined by means of a suitable computer program or, if a certain sequence is present, its probable secondary structure can be calculated.
  • Suitable vectors for the nucleic acid sequence according to the invention and in particular for the expression system according to the invention are known to those skilled in the art.
  • viral vectors are retroviruses, the RNA genome of which, after reverse transcription, is stably integrated as DNA into the genome of the host.
  • the most common are vectors based on MoMuLV used, which can only infect proliferating cells. Therefore, systems based on lentiviruses (including HIV) have also been developed, with which non-dividing cells can also be infected (review article: Miller, Hum Gene Ther. 1 (1990), 5-14; Gordon and Anderson, Curr. Opin. Biotechnol. 5 (1994), 611-616).
  • AAV adeno-associated virus
  • These ssDNA viruses are distinguished, inter alia, by the advantage that they can integrate genetic material at a defined location in chromosome 19 (review article: Grimm and Kleinschmidt, Hum. Gene. Ther. 10 (1999), 2445-2450).
  • plasmids and cosmids are used which are introduced into the cells by electroporation, lipofection or CaPO precipitation (review article: Gregoriadis, Phar. Res. 1_5 1998, 661-670).
  • An extension of this method is the use of episomal replicating plasmids which carry the "origin of replication" of the Ebstein-Barr virus (OriP) and express the EBNA-1 antigen.
  • These vectors replicate extrachromosomally in primate and dog cell lines and can be found there persist permanently (Yates et al., Nature 313 (1985), 812-815; Chittenden et al., J. Virol. 63 (1989), 3016-3025).
  • mini-chromosomes Another method of permanently replicating foreign genetic material in eukaryotic cells is the use of so-called mini-chromosomes. These large DNA molecules, like natural chromosomes, carry centromeric and telomeric sequences and are duplicated in mitosis and passed on to the daughter cells. Their size (in the megabase range) also allows very large DNA fragments of several 100,000 bases to be cloned. Mini chromosomes have been developed for yeast and mammalian cells (YACs and BACs, see: Grimes and Cooke, Hum. Mol. Genet. 7 (1998), 1635-1640; Amemiya et al, Methods Cell. Biol. 60 (1999),: 235 -258; Brown et al, Trends Biotechnol. 18 (2000), 218-223). The vectors listed here are non-limiting examples of vector systems that are described in the literature and are known to those skilled in the art.
  • the vectors are those which are suitable for the respective intended use and the cell type required or selected for this purpose.
  • the vectors are preferably those which are suitable for expression in eukaryotic cells and to control especially in mammalian cells.
  • the vectors can also be tissue-specific or control the expression of the nucleic acid ligands in a tissue-specific manner.
  • the cells according to the invention are preferably eukaryotic cells and very particularly mammalian cells.
  • the cells can be tissue-specific, undifferentiated, redifferentiated, pluripotent or stem cells.
  • the ultimate use decides the type of cells to be used.
  • the cell is to be selected depending on the tissue to be treated, which is preferably human cells and very particularly those human cells which were originally taken from the organism to which they are - again - supplied.
  • the cells can be, for example, those of mice, rats, dogs, rabbits, monkeys and humans.
  • nucleic acid sequences or expression systems is, for example, the generation of transgenic animals and plants, which is described, for example, in Dunwell, J. Exp. Bot. 51. (2000), 487-496 or. Niemann H, et al., Anim. Reprod. Be. 60-61. (2000), 277-293.
  • this application is not restricted to a specific animal or plant species, in particular not a specific mammal species, which is due to the fact that the expression and transcription machinery in eukaryotic cells is practically always constructed in the same way and functions according to the same mechanisms ,
  • the nucleic acid sequences according to the invention can be used in many areas, for example as a medicament, in target validation, in target identification, in screening programs and / or in gene therapy. Use in three-hybrid systems in eukaryotic cells for the investigation of nucleic acid / protein interactions is also conceivable (SenGupta et al., Proc. Nati. Acad. Sci. USA 93 (1996), 8496-8501). The same applies to the expression system according to the invention, the vectors according to the invention and / or the cells according to the invention.
  • the transgenic animals according to the invention can be used, for example, in the field of screening.
  • a screening program at cell culture level may have the object of targeting an antagonist to a nucleic acid ligand.
  • This could be done in a competitive assay in which a nuclear acid ligand, which is preferably expressed in a cell by means of the expression system according to the invention, influences the action of its target molecule and this influence is investigated as a function of a candidate antagonist.
  • Similar approaches can be developed for agonists and are generally known to those skilled in the art.
  • inhibitory nucleic acids could be used, for example, to inhibit enzymes and thus manipulate the content of nutrients in useful plants.
  • starch content of rice plants could be manipulated by antisense nucleic acids directed against the Wx gene and expressed in the cells (Terada et al., Plant Cell Physiol. 41 (2000), 881-888). Similar experiments could also be carried out with farm animals for the production of improved food or the provision of optimized xenografts.
  • Target validation means in particular the inactivation of a preferably cellular molecule in transgenic cells, plants or animals in which the nucleic acid ligands expressed by means of the nucleic acid sequences according to the invention bind the cellular molecule and block its function.
  • the nucleic acid ligands expressed by means of the nucleic acid sequences according to the invention bind the cellular molecule and block its function.
  • statements can then be made as to whether the cellular molecule is causally related to the phenotype under investigation, for example in a disease model.
  • These methods of reverse genetics have been successfully carried out, for example, with protein or peptide ligands against intracellular target molecules.
  • Antibodies expressed intracellularly also called intrabodies
  • an intracellular target molecule can be assigned, for example, to a phenotype associated with a specific disease, this target molecule can be used as a starting point for the development of a drug.
  • Target identification here means in particular the introduction of a combinatorial library (of different) of the nucleic acid sequences or expression systems according to the invention which ask different nucleic acid ligands inserted, preferably different nucleic acid ligands being inserted in one and the same expression system.
  • Individual nucleic acid sequences, and thus nucleic acid ligands which influence the observed phenotype can then be isolated by screening, for example, transgenic cells for the change in a particular phenotype. These individual nucleic acid sequences can then be used to isolate the intracellular target molecule whose manipulation led to a change in the phenotype and which consequently was involved in the formation of the unchanged phenotype.
  • FIG. 1 shows a schematic representation of the expression system according to the invention
  • FIG. 2 shows a comparative overview of the structure of different expression systems
  • FIG. 3 shows the secondary structures of two aptamers, namely D 28 and N3
  • FIG. 4 shows the secondary structure of D 28 which is in a pole according to the invention III-helix
  • FIG. 5 shows the secondary structure of N 3, which in a Pol III helix according to the invention Expression contract (PH1) is inserted
  • Fig. 6 shows the secondary structure of D 28, which is in an expression construct after the
  • FIG. 7 shows the secondary structure of N 3, which is inserted in an expression construct according to the prior art (Dl), FIG. 8, a total of four according to the invention designated PH1 to PH4
  • FIG. 9 a total of three according to the invention designated PH5 to PH7
  • FIG. 10 shows another expression system or construct, according to the invention, referred to as PH8, each carrying an insert, the various elements of the
  • Pol III helix expression construct polymerase III helix expression construct or polymerase III helix system are used synonymously here
  • FIG. 1 shows a schematic representation of the expression system according to the invention, which, starting with the 5 'end, comprises a Cl motif which is followed by an AI box.
  • the Al box is followed by the inserted nucleic acid, more precisely RNA, referred to as insert RNA in FIG. 1, which is a functional nucleic acid, more precisely a nucleic acid ligand, namely an aptamer.
  • the nucleic acid ligand more precisely its sequence, is followed by an A2 box, which in turn is followed by a C2 motif and an A3 box.
  • the expression construct ends at the 3 ′ terminal with a terminator.
  • a terminal double helix is formed, which defines a parent structure, at the end of which, separated by the AI box and the A2 box, the nucleic acid sequence of the nucleic acid ligand is arranged.
  • the double helix increases the stability of the construct against nuclease activity in a cellular system.
  • Targeted variations in the structure of the terminal helix allow localization of the transcripts in the cell nucleus or cytoplasm.
  • the base pairing of the 5 'end with the 3' end is important for the export of the Transcripts into the cytoplasm. For example, if 3 nucleotides of the 5 'end remain unpaired, the transcripts are retained in the cell nucleus.
  • the AI and A2 boxes are located on the 3 'side of the Cl motif and on the 5' side of the C2 motif, which preferentially comprise between 0 and 100 nucleotides. These sequence sections can contain, for example, restriction sites for cloning the insert RNA or in question promoter elements for RNA polymerases. If necessary, the A3 box can serve as a sequence spacer between the terminal helix and the terminator for the RNA polymerase in order to guarantee the structure formation of the helix. The location of the transcripts can also be controlled by the length of the A3 box. For example, transcripts with an 8-nucleotide A3 box can be exported to the cytoplasm, while transcripts with an 18-nucleotide sequence preferentially remain in the cell nucleus.
  • FIG. 2 shows a comparative overview of the structure of different expression systems, expression system A being the expression system according to the invention which contains a sequence to be expressed, referred to there as insert RNA, and contracts B to E, which are known from the prior art It should also be pointed out here that the term expression system and expression cassette are used synonymously here. To simplify matters, the expression cassettes described in the literature were consecutively labeled D1 to D4 (Fig. 2B-E).
  • the expression cassette consists of a tRNA promoter and the first three quarters of the tRNAmet in the 5 'position of the insert RNA and a stabilizing side on the 3' side
  • Expression cassette D2 tRNA expression cassette (e.g. Cotton and Birnstiel, Embo J.
  • Expression cassette is used to express RNA hybrid molecules in yeast to be expressed in the
  • RNA transcript consists of a 5 'side
  • RNaseP RNA sequence RNP leader
  • MS2 RNA sequence an MS2 RNA sequence
  • hisert RNA one Terminator for RNA polymerase III.
  • Plasmids for cloning the insert RNA and expression in the “three hybrid” system are also commercially available (for example pRH5 'or pRH3', Invitrogen BV, NV Leek, The Netherlands).
  • Expression cassette D4 lacZ expression cassette. This expression cassette was used to express aptamers against small organic molecules in eukaryotic CHO cells (Werstuck and Green, Science 282 (1998), 296-298). The DNA coding for the aptamer was cloned into the 5'-UTR of a galactosidase reporter gene in the plasmid Sv gal (Promega) and expressed via an RNA polymerase II promoter.
  • FIG. 3 shows the secondary structures of two functional nucleic acid ligands (aptamers) D 28 and N3, which are described in more detail in the examples.
  • aptamers aptamers
  • FIG. 4 shows the aptamer D28 inserted into an expression system (PH1) according to the invention.
  • the 5 'end and the 3' end of the aptamer are marked by arrows in the overall sequence. It is noteworthy that the region relevant to the binding of the nucleic acid ligand D 28, the bases of which are encircled (see also FIG. 3), also forms the required secondary structure / motif after it has been inserted into the expression system according to the invention.
  • FIG. 5 shows, analogously to FIG. 4 for the aptamer D 28, the aptamer N 3 inserted into an expression system according to the invention (PH1).
  • the 5 'end and the 3' end of the aptamer are marked by arrows in the overall sequence. It is noteworthy that the region relevant for the binding of the nucleic acid ligand N 3, the bases of which are encircled (see also FIG. 3 in this regard), also forms the required secondary structure / motif after insertion into the expression system according to the invention.
  • FIG. 6 shows the aptamer D 28 inserted into an expression construct according to the prior art (DI).
  • the expression construct is the structure D1, as shown as contract B in FIG. 2.
  • FIG. 7 shows the aptamer N3 inserted into an expression construct according to the prior art (DI).
  • the expression construct is the structure as shown as contract B in FIG. 2.
  • the expression system according to the prior art is not suitable for expressing the aptamer N 3 as functional nucleic acid in such a way that it is functionally active, since this functional activity is the presence of a certain secondary structure or of a certain motif as a prerequisite and this is not present when inserted into the expression system according to the prior art.
  • Fig. 8 Various Pol III helix expression contracts in the sense of this invention.
  • the secondary fractures of the Cl-C2 motifs with the A 1-3 boxes and the RNA polymerase III terminator are shown.
  • the frisert RNA is shown schematically.
  • the sequences of the Pol III helix expression constructs were consecutively labeled a) PH1 b) PH2 c) PH3 d) PH4).
  • PH1 has a double helix formed from the Cl motif and the C2 motif, which show a total of two mismatched pairs that are separated from one another by a range of 9 base pairs.
  • the AI box comprises 13 bases, to which the insert is shown schematically.
  • the A2 box comprises a total of 12 bases, followed by the C2 motif.
  • the A3 box only comprises one base, to which the terminator consisting of five U is connected.
  • PH 2 has a terminally located double helix formed from the Cl motif and C2 motif, which comprises only one mismatch point.
  • the AI box following the helix comprises 13 bases.
  • the A2 box comprises a total of 12 bases, followed by the C2 motif.
  • the A3 box comprises 8 bases, to which the terminator consisting of five U is connected.
  • PH 3 has a double helix that has a total of two base pairing defects that are separated by seven base pairings.
  • the AI-Box, A2-Box and A3-Box as well as the terminator are identical to the corresponding structures of PH2.
  • PH 4 also has a double helix with two base mismatches, one of the base pairing misalignments being designed such that two additional bases are present compared to the complementary sequence (U - G).
  • the AI and A2 boxes correspond to those of PH 2, and the A3 box and the terminator correspond to those of PH1.
  • FIG. 9 shows the secondary structures of further embodiments of the expression systems according to the invention, which are designated as PH5, PH 6 and PH 7.
  • the Cl. Motif has 5 'terminal three overhanging bases, which do not base pair with the bases of the C2 motif.
  • the A3 box consists of a base (C), which is followed by the terminator consisting of five U's.
  • C base
  • One of the special features of PH 5 is that the helix at 3 bases of the 5 'end is not paired and the construct thus remains in the cell nucleus.
  • the structure of the A3 box and the terminator correspond to that of PH 5.
  • a special feature of PH 6 is that the helix is paired at the 5 'end and is therefore exported to the cytoplasm.
  • PH 7 has a terminally arranged double helix with a total of four base mismatches that are set apart from one another to different extents.
  • the Cl motif comprises 34 bases and the C2 motif 31 bases.
  • the AI box comprises 14 bases and the A2 box also 14 bases.
  • the structure of the A3 box and the terminator correspond to that of PH 5.
  • the helix is paired at the 5 'end and is exported. Furthermore, the helix has an unpaired area: an internal mismatch (A: C and A: G). The helix also has internal bulges with G and UU.
  • the AI box with 5 bases is comparatively very large and forms a double helix structure both with itself and with the 7 box comprising A2 base.
  • the double helix stem formed from the 26 base Cl and the 25 bass C2 motif has a total of or three mismatch points, whereas the A3 box and the terminator correspond to those structures of PH 5.
  • the special features of PH 8 can be seen in the fact that the helix is paired at the 5 'end and the construct is thus exported.
  • the helix from the Cl and C2 motif has non-base paired areas in the form of an internal ismatch (A: C and A: G) and a bulge (G).
  • the AlBox and the A2-Box are clearly asymmetrical.
  • Mismatching sites are to be understood here as those locations in which two non-complementary bases lie opposite one another in the helix (mismatch), several non-complementary bases lie opposite one another or bases which have no bases in the complementary strand (English “bulge”).
  • Example 1 Structure of a polymerase III helix system according to the invention
  • RNA polymerase III promoter of the human U6 snRNA gene was chosen for the expression of the aptamer contracts D 28 and N3 (description see below).
  • This promoter belongs to the family of type 3 Pol-III promoters, in which all of the sequence elements acting in ice (eg a conventional TATA box or transcription factor binding sites) are located 5 'on the side of the transcription initiation site (Mattaj et al, Cell 55 (1988), 435-442; Lobo et al, Nucleic Acid Res. 18: 2891-9899 (1990)).
  • An advantage of using the U6 snRNA promoter is its extremely high transcription rate.
  • U6 snRNA per cell up to 400,000 copies of the U6 snRNA per cell are produced from only a few active U6 genes among around 200 or more pseudogenes in human cells (Hayashi, Nucleic Acid Res. 9 (1981), 3379-3389) (Weinberg et al., J. Mol. Biol. 38: 289-304 (1968).
  • RNA polymerase III promoters which are known to the person skilled in the art and are described in the literature, for example in Paule and White, Nucleic Acid Res. 28 (2000), 1283-1298), can also be used for the transcription of aptamers as long as they are compatible with the structural and functional requirements of the Pol III helix expression system.
  • the inventors developed an expression cassette (Pol III helix cassette), which is shown schematically in FIG. 1.
  • the Cl and C2 motifs form a helix between the 5 'end and the 3' end of the expression contract.
  • the Cl box and the C2 box are preferably larger than 16 base positions and form the terminal helix by at least 14 complementary Watson-Crick or non-Watson-Crick base pairs.
  • the terminal helix serves to stabilize the transcripts in the intracellular environment.
  • the transcripts can be exported to the cytoplasm or remain in the cell nucleus (see also Example 4).
  • the sequence of the nucleic acid to be expressed here the RNA, e.g. B. of the aptamer, is separated from the Cl and C2 motifs by 2 sequence sections (AI and A2 box), which are preferentially between 0 and 100 nucleotides long, and carry restriction sites for cloning the nucleic acids to be inserted or intragenous promoter elements can.
  • AI and A2 box 2 sequence sections
  • the A3 box which preferably contains 0 to 20 nucleotides.
  • the A3 box can, for example, mediate if necessary as a distance to the terminal helix and the terminator for the RNA polymerase in order to guarantee the structure formation of the helix.
  • Example 2 Preservation of the binding properties of aptamers expressed in an expression system according to the invention
  • aptamer D28 selected against the intracellular domain (CD18cyt) of the CD18 integrin subunit proteins (Blind et al, Proc. Nati. Acad. Sci. USA 96 (1999), 3606-3610).
  • the aptamer D28 was inserted into a Pol III helix expression cassette (PH1, see FIG. 4) according to the present invention and four expression cassettes described in the literature (Dl, D2, D3, D4: Good et al., Gene Ther. 4 (1997), 45-54; Cotton and Birnstiel, Embo J.
  • RNA of the different expression contracts DNA templates which additionally contain a T7 RNA polymerase promoter for in vitro transcription were produced by PCR reactions DNA templates were amplified by standard PCR, the 5 'primer containing the sequence for the T7 RNA polymerase promoter. The RNA was then produced by an in vitro transcription reaction with T7 RNA polymerase.
  • RNA of the aptamer D28 and the expression constructs, which contain the sequence of the aptamer D28 were dephosphorylated using alkaline phosphatase at the 5 'end and radioactive by kinase rank with T4 polynucleotide kinase and [32JP-ATP at the 5' end marked.
  • RNA molecules were in a volume of 20 ul binding buffer (buffer B: K 2 HPO 4 4.3 mM NaH 2 PO 4 , 1.4 mM, NaCl 150 mM, MgCl 2 1.0 mM, CaCl 2 0.1 .mu.M applied to 400 .mu.l of the target peptide derivatized CNBr-Sepharose 4B (according to the manufacturer, Pharmacia) with a column diameter of 7 mm. After the RNA molecules had been bound to the column, they were eluted again in fractions of 1000 ⁇ l by continuous washing with binding buffer.
  • buffer B K 2 HPO 4 4.3 mM NaH 2 PO 4 , 1.4 mM, NaCl 150 mM, MgCl 2 1.0 mM, CaCl 2 0.1 .mu.M
  • the radioactive fractions were measured in a scintillation counter by Cherenkov determination.
  • the elution profiles were then plotted in a diagram by adding the radioactive values (y-axis) against the corresponding elution volume (x-axis).
  • the elution profiles were used to determine the elution volume (V) at which half of the RNA molecules which had bound to the immobilized peptide (L f ) could again be eluted from the affinity matrix by fractional washing with binding buffer.
  • [Lf] concentration of the receptor immobilized on the affinity matrix (CD 18cyt).
  • V The elution volume of the RNA ligands (aptamer D28 and the different expression constructs) corresponds to the washing volume with binding buffer in which half of the RNA ligands bound to the affinity matrix were again eluted from the column.
  • V 0 total penetrable volume of the column. The elution tip of a molecule which can penetrate the matrix like the investigated CD18cyt-specific aptamers was determined on the basis of the elution volume of RNA sequences of the same length which did not bind to the peptide.
  • V m The gel-excluded volume was determined by the elution tip of a die
  • Sepharose matrix of non-penetrating molecule (dextran blue) determined.
  • K ⁇ , f dissociation constant of the ligands (aptamer D28 and the different expression constructs) to the target molecule immobilized on the matrix (peptide CD18cyt).
  • the Pol III helix construct (PH1) of the aptamer MD28 CD18cyt tested according to the invention bound with affinities comparable to those of the unchanged aptamer.
  • the dissociation constant increased drastically by at least more than an order of magnitude.
  • the inventors were able to show here that usually for the expression of e.g. Contracts used in ribozymes are not suitable for use with aptamers because they impair the binding properties.
  • the polymerase III helix system according to the invention is particularly suitable for the expression of aptamers, since the function of the molecules is only slightly impaired.
  • aptamers as functional nucleic acid molecules and especially RNA molecules, are based on the formation of a defined secondary and Tertiary structure instructed for the formation of three-dimensional interaction surfaces for their target molecules. This fact is supported by the increasing clarification of X-ray or NMR structures of the aptamer / target molecule complexes (see, for example, Zimmermann et al., Nature Struct. Biol. 4 (1997), 644-649; Rowsell et al, Nature Struct. Biol. 5: 970-974 (1998).
  • RNADrawl.l an integrated program for RNA secondary strueture calculation and analysis under 32-bit Microsoft Windows", Ole Matzura and Anders Wennborg, Computer Applications in the Biosciences (CABIOS), Vol. 12 no. 3 1996, 247-249.
  • Example 4 Extent of expression of functional nucleic acids contained in or expressed by the Pol III helix cassettes according to the invention
  • COS-1 cells were transiently transfected with the plasmid pU6 + 1 (Bertrand et al., RNA 3 (1997), 75-88) by lipofection by placing the Pol III helix cassettes through the restriction sites Sal 1 and Hind III directly behind the U6 snRNA promoter was cloned.
  • the RNA was isolated from the cells 24 h after the transfection. Total RNA was isolated by extraction using the guanidine thiocyanate-phenol-chlorofo ⁇ n method. 4 ⁇ 10 6 cells were transfected to prepare cytoplasmic RNA. After 24 h the cells were centrifuged off and washed once with cold PBS.
  • the cell pellet was placed in 400 ul Northern lysis buffer (10mM NaCl, 10mM Tris-HCl (pH 7.6), 1.5mM MgCl 2 , 5mM EDTA, pH 8.0, 0.5% NP40) for 5 min incubated on ice and the cell nuclei removed by centrifugation (14,000 rpm, 2 min, RT). 16 ⁇ l of 10% SDS and 2.5 ⁇ l of proteinase K stock solution (20 mg / ml) were added to the supernatant. After the protease treatment at 37 ° C.
  • the pellet was dissolved in 100 ⁇ l RNA buffer (150mM NaCl, 10mM Tris-Cl (pH 8.0), 1mM MgCl 2 , ImM EDTA (pH 8.0)) and with 10 U DNase 1 for 1 to remove contaminating DNA h incubated at 37 ° C. After a further phenol and chloroform fraction, the precipitated RNA was dissolved in 50 ⁇ l H 2 O.
  • RNA from the cell nucleus was isolated as follows. 10 7 cells were resuspended and washed twice with PBS (4 ° C.) and then in 7 ml of cold buffer H (15 mM NaCl, 60 mM KC1, 1 mM EDTA, 10 mM Tris, 0.2% NP40, 5% sucrose, pH 7, 5) added.
  • the cell lysates were then taken up in a cell crusher and disrupted by moving the pestle up and down four times.
  • the cell nuclei were removed by centrifugation (3500 g, 20 min) Sucrose solution (buffer H without NP40, with 10% sucrose) cleaned.
  • the RNA from the cell nuclei was then extracted by the same procedure as the total RNA.
  • RNA transcribed in vitro were immobilized as set standards on the same nylon membranes as the cellular preparations. The hybridization signals were evaluated using a phosphor imager.
  • RNA quantification of the total RNA yielded values of 2 ⁇ 10 5 to more than 4 ⁇ 10 5 copies per cell for the Pol III helix expression constructs according to the invention.
  • the number of copies was estimated on the basis of the applied amounts of cellular RNA and the signals contained therein for the respective transcripts. In comparison to other RNA expression systems described, these intracellular concentrations are very high.
  • expression levels are usually achieved which are one to two orders of magnitude below the values presented here (cf. for example Good et al., Gene Ther. 4 (1997), 45-54; Bertrand et al, RNA 3 (1997), 75-88).
  • the transcription effectiveness of the Pol III helix constructs of the invention matches that of the highly processing viral expression systems which, for example, use recombinant vaccinia viruses as vectors in conjunction with transcription units under the control of T7 RNA polymerase promoters from the T7 bacteriophage (Fuerst and Moss, J. Mol. Biol. 206 (1989), 333-348; Blind et al., Proc. Nati. Acad. Sci. USA 96 (1999), 3606-3610).
  • the Pol III helix expression system according to the invention thus surprisingly on the one hand combines very high transcription yields with structural elements which guarantee optimal functionality of aptamers.
  • RNA polymerase III promoters found in eukaryotes are suitable for the expression of functional RNA molecules in all eukaryotic cells.
  • viral systems are often limited to the respective host spectrum.
  • the cells were subjected to in situ hybridization as described in the literature (Barcellini-Couget et al., Antisense Nucleic Acid Drag Dev. 8 (1998) 379-390, Berfrand, et al, Genes Dev. 12 (1998 ), 2463-2468).
  • the specific probes for the transcripts were produced by in vitro transcription with complementary templates and at the same time labeled with digoxygenin. After hybridization, the cells were washed with 0.2X SSC, 50% formamide at 50 ° C. The signal was then detected by an anti-digoxygenin antibody coupled with alkaline phosphatase. The results were evaluated by microscopy and shown in Table 1 below. On average, approximately one hundred cells were used for each evaluation.

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Abstract

L'invention concerne une séquence d'acide nucléique pour exprimer un acide nucléique à insérer dans la séquence d'acide nucléique, ladite séquence d'acide nucléique comprenant les éléments suivants dans le sens 5'- 3' : un motif C1, une boîte A1, une boîte A2, un motif C2, une boîte A3, et un terminateur. Le motif C1 et le motif C2 forment conjointement une hélice. La boîte A1 comprend k bases, k désignant indépendamment de l un nombre entier compris entre 0 et 100 ; la boite A2 comprend l bases, l désignant indépendamment de k et de m un nombre entier compris entre 0 et 100 ; et la boîte A3 comprenant m bases, m désignant indépendamment de k et de l un nombre entier compris entre 0 et 20.
PCT/EP2001/010905 2000-09-21 2001-09-20 Systeme d'expression pour acides nucleiques fonctionnels WO2002024931A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0647716A1 (fr) * 1993-07-06 1995-04-12 Universite De Nice-Sophia Antipolis Vecteur comportant un gène viral transcrit par l'ARN polymérase III
US5695992A (en) * 1994-07-04 1997-12-09 Max Planck Gesellschaft Expression cassette for antisense expression of ribozyme

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0647716A1 (fr) * 1993-07-06 1995-04-12 Universite De Nice-Sophia Antipolis Vecteur comportant un gène viral transcrit par l'ARN polymérase III
US5695992A (en) * 1994-07-04 1997-12-09 Max Planck Gesellschaft Expression cassette for antisense expression of ribozyme

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Title
BECK J ET AL: "EFFICIENT HAMMERHEAD RIBOZYME-MEDIATED CLEAVAGE OF THE STRUCTURED HEPATITIS B VIRUS ENCAPSIDATION SIGNAL IN VITRO AND IN CELL EXTRACTS, BUT NOT IN INTACT CELLS", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 23, no. 24, 25 December 1995 (1995-12-25), pages 4954 - 4962, XP000647776, ISSN: 0305-1048 *
BERTRAND E ET AL: "THE EXPRESSION CASSETTE DETERMINES THE FUNCTIONAL ACTIVITY OF RIBOZYMES IN MAMMALIAN CELLS BY CONTROLLING THEIR INTRACELLULAR LOCALIZATION", RNA, CAMBRIDGE UNIVERSITY PRESS, CAMBRIDGE, GB, vol. 3, no. 1, 1997, pages 75 - 88, XP000646611, ISSN: 1355-8382 *
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COTTEN M ET AL: "RIBOZYME MEDIATED DESTRUCTION OF RNA IN VIVO", EMBO JOURNAL, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 8, no. 12, 1 December 1989 (1989-12-01), pages 3861 - 3866, XP000616603, ISSN: 0261-4189 *
MA YULIANG ET AL: "Structure, function, and evolution of adenovirus-associated RNA: A phylogenetic approach.", JOURNAL OF VIROLOGY, vol. 70, no. 8, 1996, pages 5083 - 5099, XP002186885, ISSN: 0022-538X *
NOONBERG S B ET AL: "IN VIVO GENERATION OF HIGHLY ABUNDANT SEQUENCE-SPECIFIC OLIGONUCLEOTIDES FOR ANTISENSE AND TRIPLEX GENE REGULATION", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 22, no. 14, 1994, pages 2830 - 2836, XP001038073, ISSN: 0305-1048 *
PERRIMAN RHONDA ET AL: "Effective ribozyme delivery in plant cells.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 92, no. 13, 1995, 1995, pages 6175 - 6179, XP002186886, ISSN: 0027-8424 *
SYMENSMA ET AL: "RNA aptamers selected to bind human immunodeficiency virus type 1 rev in vitro are rev responsive in vivo", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 70, no. 1, 1996, pages 179 - 187, XP002094212, ISSN: 0022-538X *
THOMPSON J D ET AL: "IMPROVED ACCUMULATION AND ACTIVITY OF RIBOZYMES EXPRESSED FROM A TRNA-BASED RNA POLYMERASE III PROMOTER", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 23, no. 12, 1995, pages 2259 - 2268, XP002033297, ISSN: 0305-1048 *
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