WO2001005993A1 - Cellular absorption of dna - Google Patents
Cellular absorption of dna Download PDFInfo
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- WO2001005993A1 WO2001005993A1 PCT/EP2000/006155 EP0006155W WO0105993A1 WO 2001005993 A1 WO2001005993 A1 WO 2001005993A1 EP 0006155 W EP0006155 W EP 0006155W WO 0105993 A1 WO0105993 A1 WO 0105993A1
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- Prior art keywords
- nucleic acid
- cells
- nucleic acids
- dna
- isolating
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Classifications
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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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
-
- 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
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- 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
- A61K48/0091—Purification or manufacturing processes for gene therapy compositions
-
- 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
- C12N2810/00—Vectors comprising a targeting moiety
Definitions
- the above-defined object of the present invention is achieved, in particular, in that transport signals for introducing "naked" nucleic acids, such as, for example, recombinant DNA, into cells of c / s elements, such as, for example, DNA-c / s elements, are adopted which located on the nucleic acid itself to be introduced. Therefore, according to the invention, a nucleic acid containing a nucleotide sequence with at least one signal sequence which effects the uptake of the nucleic acid into a cell is provided.
- nucleic acid and nucleotide sequence encompass endogenously expressed, semi-synthetic, synthetic or chemically modified nucleic acid molecules, which preferably consist essentially of deoxyribonucleotides and / or ribonucleotides and / or modified nucleotides.
- the “signal sequence” of the nucleic acid according to the invention is at least part of the nucleotide sequence and is thus a c / s element of the nucleic acid according to the invention and causes it to be taken up into a cell.
- the signal sequence of a DNA according to the invention is thus a DNA c / s element.
- the nucleic acid according to the invention preferably further comprises at least one second nucleotide sequence to be transported and / or one or more components which are covalently linked and / or complexed with the first and / or second nucleotide sequence and which are biologically active and are not nucleic acids.
- a second nucleotide sequence to be transported means a heterologous or homologous, recombinant nucleotide sequence which encodes, for example, one of at least one polypeptide and / or an RNA transcript of interest, for example at least one antisense nucleic acid and / or at least one ribozyme Area and / or one or more control areas and / or a partially or completely double-stranded RNA (for example an RNA in the so-called "post translational gene silencing", PTGS) and / or one or more 3
- RNA transcript of interest for example at least one antisense nucleic acid and / or at least one ribozyme Area and / or one or more control areas and / or a partially or completely double-stranded RNA (for example an RNA in the so-called "post translational gene silencing", PTGS) and / or one or more 3
- Aptamers and / or one or more nucleic acid agents for example an immunomodulatory agent such as a CpG oligonucleotide.
- region coding for at least one polypeptide means that the corresponding nucleotide sequence region has at least one open reading frame (ORF) or one or more exons, between which one or more non-translated regions (eg introns) are inserted, if appropriate, contains the or for the amino acid sequence, ie encodes or encodes the primary structure of a polypeptide.
- the region coding for at least one polypeptide can be heterologous or homologous with respect to the cell.
- region coding for at least one antisense nucleic acid means that the corresponding nucleotide sequence region codes for at least one nucleic acid which is capable of binding to a target RNA and thus its biological function, e.g.
- region coding for at least one ribozyme means that the corresponding nucleotide sequence region codes for at least one nucleic acid which is capable of cleaving a target mRNA and thus inhibiting its translation.
- a ribozyme is preferably a hammerhead ribozyme.
- control area encompasses all of gene regulation, i.e. nucleotide sequences involved, in particular in the regulation of the expression of genes, such as, for example, promoters, enhancers, operators, transcription start signals and polyadenylation signals.
- promoter denotes a nucleotide sequence section by means of which the initiation point and the initiation frequency of the transcription (mRNA synthesis) of a gene are determined.
- c / ' s active enhancer elements can develop their effect even over distances of a few kilobases. They work in both orientations and are position-independent, ie they can lie, for example, in the 5 'region of a gene controlled by them or can also lie, for example, in the 3' region. 4
- the term “operator” denotes a nucleotide sequence section to which regulatory proteins (for example repressors or activators) bind in order to thereby carry out the activities of, for example, adjacent structural genes, such as e.g. the region coding for a polypeptide, to block (negative regulation) or to activate (positive regulation).
- regulatory proteins for example repressors or activators
- transcription / translation start signal includes in particular control areas in the 5'-flanking area of nucleotide sequence sections coding for polypeptides, such as TATA and CCAT boxes, and the so-called “Kozak” located directly in the ⁇ '-flanking area of a transcription start.
- - Sequences M. Kozack (1991) "An analysis of vertebrate mRNA sequences: intimations of translational control", J. Cell Biol. 115 (4), 887-903
- ribosome binding sites and IRES elements English: “intemal ribosomal entry sites ").
- a "polyadenylation signal” denotes nucleotide sequences which are required for the addition of a poly (A) tail to an mRNA and for processing the 3 'end of the mRNA.
- the polyadenylation signals for the addition of a poly (A) tail to mammalian mRNA molecules contain two elements: the AAUAAA sequence 20 to 30 nucleiotides before and a diffuse GU-rich section directly after the 3 'end of the for the mRNA coding nucleotide sequence section.
- biologically active component encompasses all biologically active structures which consist of single or more molecules of all classes of substances with a biological function except the nucleic acids, for example peptides, e.g. Polypeptides such as proteins, lipids, e.g. Phospholipids, saccharides, e.g. individual sugars, oligosaccharides and polysaccharides are built up. Furthermore, such biologically active components can of course also contain cofactors, prosthetic groups, ions, etc.
- the control area (s) for regulating the expression of the at least one is for at least one polypeptide and / or for at least one Anti-sense nucleic acid and / or capable of coding for at least one ribozyme region.
- the expression “capable of regulating expression” means that the control area or areas control the transcription and / or translation of the at least one area coding for at least one polypeptide and / or for at least one anti-sense nucleic acid and / or for at least one ribozyme influenced or influence in a positive and / or negative manner.
- cell includes both prokaryotic cells, such as protozoa and bacteria, e.g. E.coli, Bacillus spec. And Agrobacterium tumefaciens, as well as eukaryotic cells such as fungi such as yeasts e.g. Saccharomyces cerevisiae, Schistosaccharomyces pombe and Pichia pastoris, plant cells, e.g. Tomato cells, potato cells and Arabidopsis spec, nematode cells, e.g. C. elegans, insect cells, e.g. D.
- prokaryotic cells such as protozoa and bacteria, e.g. E.coli, Bacillus spec. And Agrobacterium tumefaciens
- fungi such as yeasts e.g. Saccharomyces cerevisiae, Schistosaccharomyces pombe and Pichia pastoris
- plant cells e.g. Tomato cells, potato cells
- melanogaster and Sf9 cells as well as mammalian cells such as culture cells such as cells from endothelial, liver, muscle and brain cell lines, e.g. human culture cells (for example HeLa, ECV 304, MCF-7, A431) or culture cells of other mammals (for example BHK, CHO, PV-1) and cells which a patient suffering from a pathological disorder, for example a human or an animal, for example for Gene therapy and / or for treatment with nucleic acids and / or for in / Vo treatment.
- human culture cells for example HeLa, ECV 304, MCF-7, A431
- culture cells of other mammals for example BHK, CHO, PV-1
- pathological disorder for example a human or an animal, for example for Gene therapy and / or for treatment with nucleic acids and / or for in / Vo treatment.
- the nucleic acid according to the invention comprises one or more of the nucleotide sequences shown in FIG. 4 between the boldly marked primer sequences (SEQ ID NO 1 to 7).
- the present invention further relates to a vector which contains at least the nucleic acid as defined above.
- vector means a DNA and / or RNA replicon which can be used for the amplification and / or expression of the nucleotide sequence of the nucleic acid according to the invention.
- the vector can contain any control regions and / or other nucleotide sequence sections as defined above in the 5 'and / or 3' region of the nucleotide sequence which are capable of controlling their expression.
- the vector can additionally contain further nucleotide sequences which code for amino acid sequences which are used for the detection and / or purification of 6
- Proteins for example a polypeptide which is encoded by the nucleotide sequence according to the invention, are suitable in the 5 'and / or 3' region of the nucleotide sequence according to the invention.
- the vector according to the invention preferably contains further elements which can code the stable integration of the nucleic acid according to the invention, which, as defined above, for example for at least one polypeptide and / or at least one antisense nucleic acid and / or at least one ribozyme, into the genome of a host organism and / or enable the transient expression of the nucleotide sequence according to the invention.
- the vector according to the invention preferably contains selection genes, such as, for example, one or more antibiotic resistance genes, which enable simple selection of cells transformed with the vector according to the invention. The steps required for this are known to a person skilled in the art.
- Yet another object of the present invention relates to a host organism which contains at least the nucleic acid according to the invention or the vector according to the invention.
- the term "host organism” includes both fungi, plants, animals and humans, and parts of these, in particular their cells, such as, for example, the eukaryotic and prokaryotic cells mentioned above.
- Another object of the present invention relates to a method for producing the nucleic acid according to the invention or the vector according to the invention, comprising the steps: (a) culturing the host organism according to the invention in a suitable
- the present invention further relates to a method for identifying a nucleotide sequence which contains at least one signal sequence which causes a nucleic acid to be taken up into a cell, comprising the steps: 7
- step (a) isolating a nucleic acid that is selectively taken up by cells from a large number of nucleic acids, the large number of nucleic acids having nucleotide sequences that are at least partially different from one another, and (b) determining the nucleotide sequence of the nucleic acid isolated in step (a).
- nucleic acids encompasses endogenously expressed, semi-synthetic, synthetic or chemically modified nucleic acid molecules which have nucleotide sequences which differ at least partially from one another.
- nucleotide sequences of the large number of nucleic acids can have a variable part which differs from the variable parts of the nucleotide sequences of the other nucleic acids and an invariable part which is essentially identical in each large number of nucleic acids and can serve, for example, as a binding site for PCR primers, include.
- the origin of the nucleotide sequences of the plurality of nucleic acids is in no way limited.
- the nucleotide sequences of the multiplicity of nucleic acids can, for example, be chemically synthesized and / or originate from the genome of any biological system which contains genetic information.
- the expression “biological systems that contain genetic information” encompasses prokaryotic and eukaryotic cells as well as biological systems that cannot replicate independently, such as bacteriophages, viruses and viroids.
- the large number of nucleic acids can come from a representative library, such as, for example, a cDNA library or a genomic library, of the genome of such a biological system.
- An example of a multitude of nucleic acids comprises a DNA pool with DNA species that are, for example, 15 to several thousand base pairs in length, the randomized portion, i.e. the variable part of the nucleotide sequences of the DNA species comprises, for example, 5 to 150 base pairs and terminal primer sequences, for example of 5 to 40 base pairs in length, are added on both sides.
- the cells used for the method according to the invention can, such as 8 defined above to be all eukaryotic and prokaryotic cells.
- Preferred cells are mammalian cell lines such as BHK, Heia and ECV 304.
- isolating the nucleic acid comprises
- step (i) contacting the cells for a suitable period, for example from 1 min to 48 hours, preferably 24 hours, with the multiplicity of nucleic acids, for example a DNA pool with a sufficient complexity and in a sufficient amount, for example 1 ng to 10 ⁇ g per 10 7 cells, the cells selectively absorbing nucleic acids from the large number of nucleic acids, (ii) isolating the nucleic acid-containing constituents of the cells, (iii) detecting the nucleic acids selectively taken up by the cells from the large number of nucleic acids in the nucleic acid-containing components of the cells of (ii), and (iv) isolating the nucleic acids detected in step (iii) and selectively taken up by the cells.
- the multiplicity of nucleic acids for example a DNA pool with a sufficient complexity and in a sufficient amount, for example 1 ng to 10 ⁇ g per 10 7 cells
- the method defined above further comprises
- the detection of the nucleic acids selectively taken up by the cells in step (iii) simultaneously comprises the amplification of these nucleic acids, for example by means of amplification in living cells, for example E. coli, or by means of a PCR, preferably a preparative PCR.
- the PCR uses primers that bind, for example, to the terminal primer sequences of the DNA species as defined above in the DNA pool.
- the method according to the invention further comprises one or more washing steps, for example after the step of bringing the cells into contact with the large number of nucleic acids in step (i).
- the present invention further relates to a kit for introducing nucleic acids into cells, comprising the nucleic acid as defined above and / or the vector as defined above and / or the host organism as defined above.
- Another object of the present invention relates to a medicament containing the nucleic acid according to the invention or the vector according to the invention and optionally a pharmaceutically acceptable carrier and / or auxiliary and / or a diluent.
- the signal sequence which causes the uptake into a cell is with a region coding for at least one antisense nucleic acid and / or for at least one ribozyme and / or one or more sequence regions of nucleic acid active ingredients, for example immunomodulatory Active ingredients such as CpG oliconucleotides combined.
- the present invention thus describes a novel system for introducing nucleic acids, for example recombinant DNA or single-stranded nucleic acids, into cells, such as human cells, using novel c / s elements, for example DNA-c / s elements, which have the transport function can replace viral components or non-viral vectors previously used.
- DNA-c / s elements can, for example, introduce "naked", double-stranded or partially or completely single-stranded DNA into mammalian cells and therefore conceptually represent the simplest conceivable nucleic acid or DNA transfer into cells. plexing of recombinant DNA over viral or non-viral vector components in biomedicine are substantially supported or replaced.
- a further embodiment of the present invention therefore relates to the use of the nucleic acid according to the invention or of the vector according to the invention or of the host organism according to the invention for producing a gene therapeutic medicament.
- the nucleic acid according to the invention or the vector according to the invention or the host organism according to the invention can be used for the gene therapy treatment of patients suffering from diseases or pathological disorders which can be treated with the aid of gene therapy.
- the cellular uptake of recombinant nucleic acids via the nucleotide sequences according to the invention, which are part of the nucleic acids themselves to be introduced, means a massive simplification compared to current methods, both in technical and biological terms.
- the cellular uptake of "naked" plasmid DNA means a clear and sustainable improvement of the current state of the art.
- FIG. 1 is a schematic representation of a selection protocol according to the invention for the identification of cellular DNA import signals.
- FIG. 3 is a bar diagram which shows, by way of example, the cellular uptake of individual specific DNA species (from pool 4), the mean value of which is 48 copies per cell. The mean of the parent pool species is 7 copies per cell.
- Figure 4 shows the sequences of the seven species of Figure 3 with the best
- Uptake characteristics (clones 14, 39, 40, 59, 61, 113 and 131).
- the sequences marked in bold at the 5 'and 3' ends of the clones are the primer sequences used for amplification.
- FIG. 1 shows the selection protocol for identifying DNA signal sequences which bring about the uptake of DNA in culture cells.
- 10 ⁇ g of a DNA pool were brought into contact with cells from the BHK-21 or ECV-304 mammalian cell lines.
- the length of the DNA species was 146 base pairs, comprising a randomized portion with 100 base pairs and terminal primer sequences with 23 base pairs each.
- the complexity of the DNA pool was 10 13 different DNA molecules or species.
- the BHK-21 and ECV-304 cells (each about 10 7 cells) were incubated with the DNA pool for 24 hours. After extensive washing, the nucleic acid-containing components of the cells were isolated (intracellular fraction).
- DNA species were identified which are selectively taken up by culture cells.
- 3 shows the enrichments of individual DNA species compared to the starting pool (7 copies per cell) in a bar chart. The mean of the species shown is 48 copies per cell.
- the value of the parent pool exceeds the seven most enriched species, the nucleotide sequences of which are shown in Fig. 4, by at least eight times, while the DNA species with the greatest concentration (clone 113) also more than 36 times the concentration compared to the parent pool having.
- nucleic acid according to claim 1 wherein the nucleotide sequence further contains at least one second nucleotide sequence to be transported and / or one or more components covalently linked and / or complexed with the first and / or second nucleotide sequence, which are biologically active and are not nucleic acids.
- Nucleic acid according to claim 2, wherein the second nucleotide sequence to be transported comprises a region coding for at least one polypeptide and / or for at least one antisense nucleic acid and / or for at least one ribozyme and / or one or more control regions.
- control area (s) comprises a promoter and / or an enhancer and / or an operator and / or a transcription / translation start signal and / or a polyadenylation signal.
- control region (s) is capable of regulating the expression of the at least one region coding for at least one polypeptide and / or for at least one antisense nucleic acid and / or for at least one ribozyme ( are).
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Abstract
The invention relates to a novel system for the cellular absorption of nucleic acids, substances which contain a nucleic acid component, or genetic information. The invention relates, in particular to a nucleic acid, containing a nucleotide sequence with at least one signal sequence which causes the absorption of the nucleic acid into a cell, to a method for producing the same and to a method for identifying nucleotide sequences of this type.
Description
2 2
Diese Aufgabe wird durch die in den Ansprüchen gekennzeichneten Ausführungsformen der vorliegenden Erfindung gelöst.This object is achieved by the embodiments of the present invention characterized in the claims.
Die vorstehend definierte Aufgabe der vorliegenden Erfindung wird insbesondere dadurch gelöst, daß Transportsignale zur Einschleusung "nackter" Nukleinsäuren, wie beispielsweise rekombinanter DNA, in Zellen von c/s-Elementen, wie beispielsweise DNA-c/s-Elementen, übernommen werden, die sich auf der einzuschleusenden Nukleinsäure selbst befinden. Daher wird erfindungsgemäß eine Nukleinsäure, enthaltend eine Nukleotidsequenz mit mindestens einer Signalsequenz, welche die Aufnahme der Nukleinsäure in eine Zelle bewirkt, bereitgestellt.The above-defined object of the present invention is achieved, in particular, in that transport signals for introducing "naked" nucleic acids, such as, for example, recombinant DNA, into cells of c / s elements, such as, for example, DNA-c / s elements, are adopted which located on the nucleic acid itself to be introduced. Therefore, according to the invention, a nucleic acid containing a nucleotide sequence with at least one signal sequence which effects the uptake of the nucleic acid into a cell is provided.
Die Begriffe "Nukleinsäure" und "Nukleotidsequenz" umfassen endogen ex- primierte, halbsynthetische, synthetische oder chemisch modifizierte Nukleinsäu- remoleküle, die vorzugsweise im wesentlichen aus Desoxyribonukleotiden und/oder Ribonukleotiden und/oder modifizierten Nukleotiden bestehen. Die "Signalsequenz" der erfindungsgemäßen Nukleinsäure ist mindestens ein Teil der Nukleotidsequenz und ist somit ein c/s-Element der erfindungsgemäßen Nukleinsäure und bewirkt deren Aufnahme in eine Zelle. Beispielsweise handelt es sich bei der Signalsequenz einer erfindungsgemäßen DNA somit um ein DNA-c/s- Element.The terms “nucleic acid” and “nucleotide sequence” encompass endogenously expressed, semi-synthetic, synthetic or chemically modified nucleic acid molecules, which preferably consist essentially of deoxyribonucleotides and / or ribonucleotides and / or modified nucleotides. The “signal sequence” of the nucleic acid according to the invention is at least part of the nucleotide sequence and is thus a c / s element of the nucleic acid according to the invention and causes it to be taken up into a cell. For example, the signal sequence of a DNA according to the invention is thus a DNA c / s element.
Vorzugsweise enthält die erfindungsgemäße Nukleinsäure weiter mindestens eine zu transportierende zweite Nukleotidsequenz und/oder eine oder mehrere mit der ersten und/oder zweiten Nukleotidsequenz kovalent verknüpfte und/oder komplexierte Komponenten, die biologisch aktiv und keine Nukleinsäuren sind.The nucleic acid according to the invention preferably further comprises at least one second nucleotide sequence to be transported and / or one or more components which are covalently linked and / or complexed with the first and / or second nucleotide sequence and which are biologically active and are not nucleic acids.
Der Ausdruck "eine zu transportierende zweite Nukleotidsequenz" bedeutet eine heterologe oder homologe, rekombinante Nukleotidsequenz, die beispielsweise einen für mindestens ein Polypeptid und/oder ein RNA-Transkript von Interesse, z.B. für mindestens eine Antisense-Nukleinsäure und/oder für mindestens ein Ribozym kodierenden Bereich und/oder einen oder mehrere Kontrollbereiche und/der eine partiell oder vollständig doppelsträngige RNA (z.B. eine RNA beim sog. "post translational gene silencing", PTGS) und/oder einen oder mehrere
3The expression "a second nucleotide sequence to be transported" means a heterologous or homologous, recombinant nucleotide sequence which encodes, for example, one of at least one polypeptide and / or an RNA transcript of interest, for example at least one antisense nucleic acid and / or at least one ribozyme Area and / or one or more control areas and / or a partially or completely double-stranded RNA (for example an RNA in the so-called "post translational gene silencing", PTGS) and / or one or more 3
Aptamere und/oder einen oder mehrere Nukleinsäure-Wirkstoffe, beispielsweise einen immunmodulatorischen Wirkstoff wie ein CpG-Oligonukleotid, umfassen kann.Aptamers and / or one or more nucleic acid agents, for example an immunomodulatory agent such as a CpG oligonucleotide.
Der Ausdruck "für mindestens ein Polypeptid kodierender Bereich" bedeutet, daß der entsprechende Nukleotidsequenzbereich mindestens einen offenen Leserahmen (ORF) bzw. ein oder mehrere Exons, zwischen denen gegebenenfalls ein oder mehrere nicht-translatierte Bereiche (z. B. Introns) eingefügt sind, enthält, der bzw. die für die Aminosäuresequenz, d.h. die Primärstruktur, eines Polypep- tids kodiert bzw. kodieren. Der mindestens für ein Polypeptid kodierende Bereich kann dabei in Bezug auf die Zelle heterolog oder homolog sein. Der Ausdruck "für mindestens eine Antisense-Nukleinsäure kodierender Bereich" bedeutet, daß der entsprechende Nukleotidsequenzbereich mindestens für eine Nukleinsäure kodiert, die zur Bindung an eine Ziel-RNA befähigt ist und so deren biologische Funktion, z.B. die Translation einer mRNA inhibiert. Der Ausdruck "für mindestens ein Ribozym kodierender Bereich" bedeutet, daß der entsprechende Nukleotidsequenzbereich mindestens für eine Nukleinsäure, kodiert, die zur Spaltung einer Ziel-mRNA befähigt ist und so deren Translation inhibiert. Vorzugsweise handelt es sich bei einem solchen Ribozym um ein Hammerhead-Ribozym.The expression “region coding for at least one polypeptide” means that the corresponding nucleotide sequence region has at least one open reading frame (ORF) or one or more exons, between which one or more non-translated regions (eg introns) are inserted, if appropriate, contains the or for the amino acid sequence, ie encodes or encodes the primary structure of a polypeptide. The region coding for at least one polypeptide can be heterologous or homologous with respect to the cell. The term "region coding for at least one antisense nucleic acid" means that the corresponding nucleotide sequence region codes for at least one nucleic acid which is capable of binding to a target RNA and thus its biological function, e.g. inhibits the translation of an mRNA. The expression “region coding for at least one ribozyme” means that the corresponding nucleotide sequence region codes for at least one nucleic acid which is capable of cleaving a target mRNA and thus inhibiting its translation. Such a ribozyme is preferably a hammerhead ribozyme.
Der Begriff "Kontrollbereich" umfaßt sämtliche bei der Genregulation, d.h. insbesondere bei der Regulation der Expression von Genen, mitwirkende Nukleo- tidsequzenzen, wie beispielsweise Promotoren, Enhancer, Operatoren, Transkriptionsstartsignale und Polyadenylierungssignale. Der Begriff "Promotor" bezeichnet einen Nukleotidsequenzabschnitt, durch den der Initiationspunkt und die Initiationshäufigkeit der Transkription (mRNA-Syn- these) eines Gens festgelegt werden.The term "control area" encompasses all of gene regulation, i.e. nucleotide sequences involved, in particular in the regulation of the expression of genes, such as, for example, promoters, enhancers, operators, transcription start signals and polyadenylation signals. The term “promoter” denotes a nucleotide sequence section by means of which the initiation point and the initiation frequency of the transcription (mRNA synthesis) of a gene are determined.
Der Begriff "Enhancer" bezeichnet Nukleotidsequenzabschnitte, wie sie beispielsweise im 5'- oder 3'-flankierenden Bereich von Genen, insbesondere solcher eukaryotischer und viraler Herkunft zu finden sind, welche die Effizienz der Transkription erhöhen. Die c/'s-aktiven Enhancer-Elemente können selbst über Distanzen von einigen Kilobasen ihre Wirkung entfalten. Sie funktionieren in beiden Orientierungen und sind positionsunabhängig, d.h., sie können z.B. im 5'- Bereich eines durch sie kontrollierten Gens liegen oder können auch z.B. im 3'-
4The term “enhancer” refers to nucleotide sequence segments, such as those found in the 5'- or 3'-flanking region of genes, in particular those of eukaryotic and viral origin, which increase the efficiency of the transcription. The c / ' s active enhancer elements can develop their effect even over distances of a few kilobases. They work in both orientations and are position-independent, ie they can lie, for example, in the 5 'region of a gene controlled by them or can also lie, for example, in the 3' region. 4
Bereich oder sogar beispielsweise in einem Intron lokalisiert sein. Der Begriff "Operator" bezeichnet in der vorliegenden Erfindung einen Nukleotidsequenzabschnitt, an dem regulatorisch wirkende Proteine (beispielsweise Repressoren oder Aktivatoren) binden, um dadurch die Aktivitäten von beispiels- weise angrenzenden Strukturgenen, wie z.B. des für ein Polypeptid kodierenden Bereichs, zu blockieren (negative Regulation) oder zu aktivieren (positive Regulation).Area or even localized, for example, in an intron. In the present invention, the term “operator” denotes a nucleotide sequence section to which regulatory proteins (for example repressors or activators) bind in order to thereby carry out the activities of, for example, adjacent structural genes, such as e.g. the region coding for a polypeptide, to block (negative regulation) or to activate (positive regulation).
Der Begriff "Transkriptions/Translationsstartsignal" umfaßt insbesondere Kontrollbereiche im 5'-flankierenden Bereich von für Polypeptide kodierenden Nukleo- tidsequenzabschnitten, wie beispielsweise TATA- und CCAT-Boxen, sowie die unmittelbar im δ'-flankierenden Bereich eines Transkriptionsstarts befindlichen sog. "Kozak"-Sequenzen (M. Kozack (1991) "An analysis of vertebrate mRNA sequences: intimations of translational control", J. Cell Biol. 115(4), 887-903) sowie Ribosomenbindungsstellen und IRES-Elemente (engl. "intemal ribosomal entry Sites").The term "transcription / translation start signal" includes in particular control areas in the 5'-flanking area of nucleotide sequence sections coding for polypeptides, such as TATA and CCAT boxes, and the so-called "Kozak" located directly in the δ'-flanking area of a transcription start. - Sequences (M. Kozack (1991) "An analysis of vertebrate mRNA sequences: intimations of translational control", J. Cell Biol. 115 (4), 887-903) as well as ribosome binding sites and IRES elements (English: "intemal ribosomal entry sites ").
Ein "Polyadenylierungssignal" bezeichnet Nukleotidsquenzen, die für die Addition eines Poly(A)-Schwanzes an eine mRNA und zur Prozessierung des 3'-Endes der mRNA benötigt werden. Die Polyadenylierungssignale für die Addition eines poly(A)-Schwanzes an mRNA-Moleküle von Säugern enthalten beispielsweise zwei Elemente: Die AAUAAA-Sequenz 20 bis 30 Nukleiotide vor und einen diffusen GU-reichen Abschnitt direkt nach dem 3'-Ende des für die mRNA kodierenden Nukleotidsequenzabschnitts.A "polyadenylation signal" denotes nucleotide sequences which are required for the addition of a poly (A) tail to an mRNA and for processing the 3 'end of the mRNA. For example, the polyadenylation signals for the addition of a poly (A) tail to mammalian mRNA molecules contain two elements: the AAUAAA sequence 20 to 30 nucleiotides before and a diffuse GU-rich section directly after the 3 'end of the for the mRNA coding nucleotide sequence section.
Der Begriff "biologisch aktive Komponente" umfaßt alle biologisch aktiven Strukturen, die aus einzelnen oder mehreren Molekülen sämtlicher Substanzklassen mit biologischer Funktion außer den Nukleinsäuren, beispielsweise Peptide, z.B. Polypeptide, wie Proteine, Lipide, z.B. Phospholipide, Saccharide, z.B. einzelne Zucker, Oligo- und Polysaccharide aufgebaut sind. Des weiteren können solche biologisch aktiven Komponenten selbstverständlich auch Cofakto- ren, prosthetische Gruppen, Ionen usw. enthalten.The term "biologically active component" encompasses all biologically active structures which consist of single or more molecules of all classes of substances with a biological function except the nucleic acids, for example peptides, e.g. Polypeptides such as proteins, lipids, e.g. Phospholipids, saccharides, e.g. individual sugars, oligosaccharides and polysaccharides are built up. Furthermore, such biologically active components can of course also contain cofactors, prosthetic groups, ions, etc.
Gemäß einer bevorzugten Ausführungsform der erfindungsgemäßen Nukleinsäure ist (sind) der (die) Kontrollbereich(e) zur Regulation der Expression des mindestens einen für mindestens ein Polypeptid und/oder für mindestens eine
Anti-sense-Nukleinsäure und/oder für mindestens ein Ribozym kodierenden Bereichs befähigt. Der Ausdruck "zur Regulation der Expression befähigt" bedeutet, daß der bzw. die Kontrollbereiche die Transkription und/oder Translation des mindestens einen für mindestens ein Polypeptid und/oder für mindestens eine Anti-sense-Nukleinsäure und/oder für mindestens ein Ribozym kodierenden Bereichs in positiver und/oder negativer Weise beeinflußt bzw. beeinflussen.According to a preferred embodiment of the nucleic acid according to the invention, the control area (s) for regulating the expression of the at least one is for at least one polypeptide and / or for at least one Anti-sense nucleic acid and / or capable of coding for at least one ribozyme region. The expression “capable of regulating expression” means that the control area or areas control the transcription and / or translation of the at least one area coding for at least one polypeptide and / or for at least one anti-sense nucleic acid and / or for at least one ribozyme influenced or influence in a positive and / or negative manner.
Der Begriff "Zelle" umfaßt sowohl prokaryotische Zellen, wie beispiesweise Protozoen und Bakterien, z.B. E.coli, Bacillus spec.und Agrobacterium tumefa- ciens, als auch eukaryotische Zellen, wie beispielsweise Pilze, wie Hefen, z.B. Saccharomyces cerevisiae, Schistosaccharomyces pombe und Pichia pastoris, Pflanzenzellen, z.B. Tomatenzellen, Kartoffelzellen und Arabidopsis spec, Nematodenzellen, z.B. C. elegans, Insektenzellen, z.B. D. melanogaster und Sf9- Zellen, sowie Säugerzellen, wie beispielsweise Kulturzellen wie Zellen von Endothel-, Leber-, Muskel- und Hirnzellinien, z.B. humane Kulturzellen (z.B. HeLa, ECV 304, MCF-7, A431 ) oder Kulturzellen anderer Säuger (z.B. BHK, CHO, PV-1 ) sowie Zellen, die einem an einer krankhaften Störung leidenden Patienten, beispielsweise einem Menschen oder einem Tier, beispielsweise zur Gentherapie und/oder zur Behandlung mit Nukleinsäuren und/oder zur in /Vo-Behandlung, entnommen wurden.The term "cell" includes both prokaryotic cells, such as protozoa and bacteria, e.g. E.coli, Bacillus spec. And Agrobacterium tumefaciens, as well as eukaryotic cells such as fungi such as yeasts e.g. Saccharomyces cerevisiae, Schistosaccharomyces pombe and Pichia pastoris, plant cells, e.g. Tomato cells, potato cells and Arabidopsis spec, nematode cells, e.g. C. elegans, insect cells, e.g. D. melanogaster and Sf9 cells, as well as mammalian cells such as culture cells such as cells from endothelial, liver, muscle and brain cell lines, e.g. human culture cells (for example HeLa, ECV 304, MCF-7, A431) or culture cells of other mammals (for example BHK, CHO, PV-1) and cells which a patient suffering from a pathological disorder, for example a human or an animal, for example for Gene therapy and / or for treatment with nucleic acids and / or for in / Vo treatment.
In einer bevorzugten Ausführungsform umfaßt die erfindungsgemäße Nukleinsäure eine oder mehrere der in der Fig. 4 zwischen den fett markierten Primersequenzen gezeigten Nukleotidsequenzen (SEQ ID NO 1 bis 7).In a preferred embodiment, the nucleic acid according to the invention comprises one or more of the nucleotide sequences shown in FIG. 4 between the boldly marked primer sequences (SEQ ID NO 1 to 7).
Ein weiterer Gegenstand der vorliegenden Erfindung betrifft einen Vektor, der mindestens die wie vorstehend definierte Nukleinsäure enthält. Der Begriff "Vektor" bedeutet dabei ein DNA- und/oder RNA-Replikon, das zur Amplifikation und/oder Expression der Nukleotidsequenz der erfindungsgemäßen Nukleinsäure verwendet werden kann. Der Vektor kann jegliche wie vorstehend definierte Kontrollbereiche und/oder andere Nukeotidsequenzabschnitte im 5'- und/oder 3'- Bereich der Nukleotidsequenz, die zur Kontrolle ihrer Expression befähigt sind, enthalten. Der Vektor kann zusätzlich weitere Nukleotidsequenzen, die für Aminosäuresequenzen kodieren, die zur Detektion und/oder Reinigung von
6The present invention further relates to a vector which contains at least the nucleic acid as defined above. The term "vector" means a DNA and / or RNA replicon which can be used for the amplification and / or expression of the nucleotide sequence of the nucleic acid according to the invention. The vector can contain any control regions and / or other nucleotide sequence sections as defined above in the 5 'and / or 3' region of the nucleotide sequence which are capable of controlling their expression. The vector can additionally contain further nucleotide sequences which code for amino acid sequences which are used for the detection and / or purification of 6
Proteinen, beipielsweise eines Polypeptids, das von der erfindungsgemäßen Nukleotidsequenz kodiert wird, geeignet sind, im 5'- und/oder 3'-Bereich der erfindungsgemäßen Nukleotidsequenz enthalten. Vorzugsweise enthält der erfindungsgemäße Vektor weitere Elemente, welche die stabile Integration der erfindungsgemäßen Nukleinsäure, die wie vorstehend definiert beispielsweise für mindestens ein Polypeptid und/oder mindestens eine Antisense-Nukleinsäure und/oder mindestens ein Ribozym kodieren kann, in das Genom eines Wirtsorganismus und/oder die transiente Expression der erfindungsgemäßen Nukleotidsequenz ermöglichen. Ferner enthält der erfindungsgemäße Vektor vorzugs- weise Selektionsgene, wie beispielsweise ein oder mehrere Antibiotikaresi- stenzgene, welche die einfache Selektion mit dem erfindungsgemäßen Vektor transformierter Zellen ermöglichen. Die hierfür notwendigen Arbeitsschritte sind einem Fachmann bekannt.Proteins, for example a polypeptide which is encoded by the nucleotide sequence according to the invention, are suitable in the 5 'and / or 3' region of the nucleotide sequence according to the invention. The vector according to the invention preferably contains further elements which can code the stable integration of the nucleic acid according to the invention, which, as defined above, for example for at least one polypeptide and / or at least one antisense nucleic acid and / or at least one ribozyme, into the genome of a host organism and / or enable the transient expression of the nucleotide sequence according to the invention. Furthermore, the vector according to the invention preferably contains selection genes, such as, for example, one or more antibiotic resistance genes, which enable simple selection of cells transformed with the vector according to the invention. The steps required for this are known to a person skilled in the art.
Noch ein weiterer Gegenstand der vorliegenden Erfindung betrifft einen Wirtsorganismus, der mindestens die erfindungsgemäße Nukleinsäure oder den erfindungsgemäßen Vektor enthält. Der Begriff "Wirtsorganismus" umfaßt sowohl Pilze, Pflanzen, Tiere und Menschen, als auch Teile dieser, insbesondere deren Zellen, wie beispielsweise die vorstehend angebenen eukaryotischen und prokaryotischen Zellen.Yet another object of the present invention relates to a host organism which contains at least the nucleic acid according to the invention or the vector according to the invention. The term "host organism" includes both fungi, plants, animals and humans, and parts of these, in particular their cells, such as, for example, the eukaryotic and prokaryotic cells mentioned above.
Ein weiterer Gegenstand der vorliegenden Erfindung betrifft ein Verfahren zur Herstellung der erfindungsgemäßen Nukleinsäure oder des erfindungsge-mäßen Vektors, umfassend die Schritte: (a) Züchten des erfindungsgemäßen Wirtsorganismus in einem geeignetenAnother object of the present invention relates to a method for producing the nucleic acid according to the invention or the vector according to the invention, comprising the steps: (a) culturing the host organism according to the invention in a suitable
Medium unter geeigneten Bedingungen und (b) Isolieren des gewünschten Produkts aus dem Medium und/oder demMedium under suitable conditions and (b) isolating the desired product from the medium and / or the
Wirtsorganismus.Host organism.
Ein weiterer Gegenstand der vorliegenden Erfindung betrifft ein Verfahren zur Identifizierung einer Nukleotidsequenz, die mindestens eine die Aufnahme einer Nukleinsäure in eine Zelle bewirkende Signalsequenz enthält, umfassend die Schritte:
7The present invention further relates to a method for identifying a nucleotide sequence which contains at least one signal sequence which causes a nucleic acid to be taken up into a cell, comprising the steps: 7
(a) Isolieren einer Nukleinsäure, die von Zellen selektiv aus einer Vielzahl von Nukleinsäuren aufgenommen wird, wobei die Vielzahl der Nukleinsäuren Nukleotidsequenzen aufweisen, die sich mindestens teilweise voneinander unterscheiden, und (b) Bestimmen der Nukleotidsequenz der in Schritt (a) isolierten Nukleinsäure.(a) isolating a nucleic acid that is selectively taken up by cells from a large number of nucleic acids, the large number of nucleic acids having nucleotide sequences that are at least partially different from one another, and (b) determining the nucleotide sequence of the nucleic acid isolated in step (a).
Der Begriff "Viehlzahl von Nukleinsäuren" umfaßt endogen exprimierte, halbsynthetische, synthetische oder chemische modifizierte Nukleinsäuremoleküle, die Nukleotidsequenzen aufweisen, die sich mindestens teilweise voneinander unterscheiden. Beispielsweise können die Nukleotidsequenzen der Vielzahl von Nukleinsäuren einen variablen Teil, der sich von den variablen Teilen der Nukleotidsequenzen der anderen Nukleinsäuren unterscheidet und einen invariablen Teil, der in jeder Vielzahl von Nukleinsäuren im wesentlichen identisch ist und beispielsweise als Bindungsstelle von PCR-Primem dienen kann, umfassen. Wie vorstehend definiert, ist die Herkunft der Nukleotidsequenzen der Vielzahl der Nukleinsäuren in keiner Weise begrenzt. Die Nukleotidsequenzen der Vielzahl von Nukleinsäuren können beispielsweise chemisch synthetisiert sein, und/oder von dem Genom jeglicher biologischer Systeme, die genetische Information enthalten, stammen. Der Ausdruck "biologische System, die genetische Information enthalten" umfaßt dabei sowohl prokaryotische und eukaryotische Zellen als auch biologische Systeme, die sich nicht selbständig replizieren können, wie Bakteriophagen, Viren und Viroide. So kann die Vielzahl der Nukleinsäuren beispielsweise von einer representativen Bibliothek, wie beispielsweise einer cDNA-Bibliothek oder einer genomischen Bibliothek, des Genoms eines solchen biologischen Systems stammen.The term "multitude of nucleic acids" encompasses endogenously expressed, semi-synthetic, synthetic or chemically modified nucleic acid molecules which have nucleotide sequences which differ at least partially from one another. For example, the nucleotide sequences of the large number of nucleic acids can have a variable part which differs from the variable parts of the nucleotide sequences of the other nucleic acids and an invariable part which is essentially identical in each large number of nucleic acids and can serve, for example, as a binding site for PCR primers, include. As defined above, the origin of the nucleotide sequences of the plurality of nucleic acids is in no way limited. The nucleotide sequences of the multiplicity of nucleic acids can, for example, be chemically synthesized and / or originate from the genome of any biological system which contains genetic information. The expression “biological systems that contain genetic information” encompasses prokaryotic and eukaryotic cells as well as biological systems that cannot replicate independently, such as bacteriophages, viruses and viroids. For example, the large number of nucleic acids can come from a representative library, such as, for example, a cDNA library or a genomic library, of the genome of such a biological system.
Ein Beispiel einer Vielzahl von Nukleinsäuren umfaßt einen DNA-Pool mit DNA- Spezies, die beispielsweise eine Länge von 15 bis mehreren tausend Basenpaaren aufweisen, wobei der randomisierte Anteil, d.h. der variable Teil der Nukleo- tidsequenzen der DNA-Spezies, beispielsweise 5 bis 150 Basenpaare umfaßt und beidseitig terminale Primersequenzen, beispielsweise von 5 bis 40 Basenpaare Länge, angefügt sind.An example of a multitude of nucleic acids comprises a DNA pool with DNA species that are, for example, 15 to several thousand base pairs in length, the randomized portion, i.e. the variable part of the nucleotide sequences of the DNA species comprises, for example, 5 to 150 base pairs and terminal primer sequences, for example of 5 to 40 base pairs in length, are added on both sides.
Die für das erfindungsgemäße Verfahren verwendeten Zellen können, wie
8 vorstehend definiert, sämtliche eukaryotische und prokaryotische Zellen sein. Bevorzugte Zellen sind Säugerzellinien, wie beispielsweise BHK, Heia und ECV 304.The cells used for the method according to the invention can, such as 8 defined above to be all eukaryotic and prokaryotic cells. Preferred cells are mammalian cell lines such as BHK, Heia and ECV 304.
Gemäß einer bevorzugten Ausführungsform des vorstehend definierten Verfahrens umfaßt das Isolieren der NukleinsäureAccording to a preferred embodiment of the method defined above, isolating the nucleic acid comprises
(i) das Inkontaktbringen der Zellen für einen geeigneten Zeitraum, beispielsweise von 1 min bis 48 Stunden, vorzugsweise 24 Stunden, mit der Vielzahl von Nukleinsäuren, beispielsweise einem DNA-Pool mit einer ausreichenden Komplexität und in einer ausreichenden Menge, beispielsweise 1 ng bis 10 μg pro 107 Zellen, wobei die Zellen selektiv Nukleinsäuren aus der Vielzahl von Nukleinsäuren aufnehmen, (ii) das Isolieren der Nukleinsäure-haltigen Bestandteile der Zellen, (iii) das Detektieren der von den Zellen selektiv aus der Vielzahl der Nuklein- säuren aufgenommenen Nukleinsäuren in den Nukleinsäure-haltigen Bestandteilen der Zellen von (ii), und (iv) das Isolieren der im Schritt (iii) detektierten, selektiv von den Zellen aufgenommenen Nukleinsäuren.(i) contacting the cells for a suitable period, for example from 1 min to 48 hours, preferably 24 hours, with the multiplicity of nucleic acids, for example a DNA pool with a sufficient complexity and in a sufficient amount, for example 1 ng to 10 μg per 10 7 cells, the cells selectively absorbing nucleic acids from the large number of nucleic acids, (ii) isolating the nucleic acid-containing constituents of the cells, (iii) detecting the nucleic acids selectively taken up by the cells from the large number of nucleic acids in the nucleic acid-containing components of the cells of (ii), and (iv) isolating the nucleic acids detected in step (iii) and selectively taken up by the cells.
Gemäß einer bevorzugten Ausführungsform umfaßt das vorstehend definierte Verfahren weiterAccording to a preferred embodiment, the method defined above further comprises
(v) das Wiederholen der Schritte (i) bis (iv), bis sich der Anteil der aus der(v) repeating steps (i) through (iv) until the proportion of the
Vielzahl von Nukleinsäuren selektiv aufgenommenen Nukleinsäuren an denVariety of nucleic acids selectively added to the
Nukleinsäure-haltigen Bestandteilen der Zellen nicht mehr ändert, wobei jeweils im Schritt (i) die im Schritt (iv) isolierten Nukleinsäuren mit denNucleic acid-containing components of the cells no longer change, with the nucleic acids isolated in step (iv) in each case in step (i)
Zellen in Kontakt gebracht werden.Cells are brought into contact.
Vorzugsweise umfaßt das Detektieren der von den Zellen selektiv aufgenommenen Nukleinsäuren im Schritt (iii) gleichzeitig die Amplifikation dieser Nukleinsäu- ren, beispielsweise mittels Amplifikation in lebenden Zellen, z.B. E. coli, oder mittels einer PCR, vorzugsweise einer präparativen PCR. Dabei werden bei der PCR Primer eingesetzt, die beispielsweise an die terminalen Primersequenzen der wie vorstehend definierten DNA-Spezies im DNA-Pool binden. Ferner umfaßt das erfindungsgemäße Verfahren in einer bevorzugten Ausführungsform weiter
ein oder mehrere Waschschritte, beispielsweise nach dem Schritt des Inkontakt- bringens der Zellen mit der Vielzahl von Nukleinsäuren im Schritt (i).Preferably, the detection of the nucleic acids selectively taken up by the cells in step (iii) simultaneously comprises the amplification of these nucleic acids, for example by means of amplification in living cells, for example E. coli, or by means of a PCR, preferably a preparative PCR. The PCR uses primers that bind, for example, to the terminal primer sequences of the DNA species as defined above in the DNA pool. Furthermore, in a preferred embodiment, the method according to the invention further comprises one or more washing steps, for example after the step of bringing the cells into contact with the large number of nucleic acids in step (i).
Ein weiterer Gegenstand der vorliegenden Erfindung betrifft ein Kit zur Ein- Schleusung von Nukleinsäuren in Zellen, umfasend die wie vorstehend definierte Nukleinsäure und/oder den wie vorstehend definierten Vektor und/oder den wie vorstehend definierten Wirtsorganismus.The present invention further relates to a kit for introducing nucleic acids into cells, comprising the nucleic acid as defined above and / or the vector as defined above and / or the host organism as defined above.
Ein weiterer Gegenstand der vorliegenden Erfindung betrifft ein Arzneimittel, enthaltend die erfindungsgemäße Nukleinsäure oder den erfindungsgemäßen Vektor und gegebenenfalls einen pharmazeutisch verträglichen Träger und/oder Hilfsstoff und/oder ein Verdünnungsmittel. In bevorzugten Beispielen des erfindungsgemäßen Arzneimittels ist die Signalsequenz, welche die Aufnahme in eine Zelle bewirkt, mit einem für mindestens eine Antisense-Nukleinsäure und/oder für mindestens ein Ribozym kodierenden Bereich und/oder einem oder mehreren Sequenzbereichen von Nukleinsäure-Wirkstoffen, beispielsweise von immunmodulatorischen Wirkstoffen wie CpG-Olikonukleotiden, kombiniert.Another object of the present invention relates to a medicament containing the nucleic acid according to the invention or the vector according to the invention and optionally a pharmaceutically acceptable carrier and / or auxiliary and / or a diluent. In preferred examples of the medicament according to the invention, the signal sequence which causes the uptake into a cell is with a region coding for at least one antisense nucleic acid and / or for at least one ribozyme and / or one or more sequence regions of nucleic acid active ingredients, for example immunomodulatory Active ingredients such as CpG oliconucleotides combined.
Die vorliegende Erfindung beschreibt somit ein neuartiges System zur Einschleu- sung von Nukleinsäuren, beispielsweise rekombinanter DNA oder einzelsträngiger Nukleinsäuren, in Zellen, wie beispielsweise menschliche Zellen, über neuartige c/s-Elemente, beispielsweise DNA-c/s-Elemente, welche die Transportfunktion von bisher benutzten viralen Komponenten oder nicht-viralen Vektoren ersetzen können. Solche DNA-c/s-Elemente können beispielsweise "nackte", doppel- strängige oder partiell oder vollständig einzelsträngige DNA in Säugerzellen einschleusen und stellen daher konzeptionell den einfachsten denkbaren Nukleinsäure- bzw. DNA-Transfer in Zellen dar. Damit kann die aufwendige Kom- plexierung rekombinanter DNA über virale oder nicht-virale Vektorkomponenten in der Biomedizin substantiell unterstützt oder ersetzt werden. Es ist ebenfalls möglich, daß rekombinante Gene von Interesse in der molekularen Medizin, wie beispielsweise solcher in der Gentherapie, mit zusätzlichen erfindungsgemäßen DNA-Transportelementen versehen werden und so direkt einsetzbar sind, was eine drastische Vereinfachung von Protokollen in der somatischen Gentherapie zur Folgen hat.
Daher betrifft eine weitere Ausführungsform der vorliegenden Erfindung die Verwendung der erfindungsgemäßen Nukleinsäure oder des erfindungsgemäßen Vektors oder des erfindungsgemäßen Wirtsorganismus zur Herstellung eines gentherapeutischen Arzneimittels. Zum Beispiel kann die erfindungsgemäße Nukleinsäure oder der erfindungsgemäße Vektor oder der erfindungsgemäße Wirtsorganismus zur gentherapeutischen Behandlung von Patienten, die an Krankheiten bzw. krankhaften Störungen leiden, die mit Hilfe der Gentherapie behandelt werden können, verwendet werden.The present invention thus describes a novel system for introducing nucleic acids, for example recombinant DNA or single-stranded nucleic acids, into cells, such as human cells, using novel c / s elements, for example DNA-c / s elements, which have the transport function can replace viral components or non-viral vectors previously used. Such DNA-c / s elements can, for example, introduce "naked", double-stranded or partially or completely single-stranded DNA into mammalian cells and therefore conceptually represent the simplest conceivable nucleic acid or DNA transfer into cells. plexing of recombinant DNA over viral or non-viral vector components in biomedicine are substantially supported or replaced. It is also possible that recombinant genes of interest in molecular medicine, such as those in gene therapy, are provided with additional DNA transport elements according to the invention and can thus be used directly, which has the consequence of a drastic simplification of protocols in somatic gene therapy. A further embodiment of the present invention therefore relates to the use of the nucleic acid according to the invention or of the vector according to the invention or of the host organism according to the invention for producing a gene therapeutic medicament. For example, the nucleic acid according to the invention or the vector according to the invention or the host organism according to the invention can be used for the gene therapy treatment of patients suffering from diseases or pathological disorders which can be treated with the aid of gene therapy.
Die zelluläre Aufnahme rekombinanter Nukleinsäuren über die erfindungsgemäßen Nukleotidsequenzen, die Teil der einzuschleusenden Nukleinsäuren selbst sind, bedeutet sowohl in technischer als auch in biologischer Sicht eine massive Vereinfachung gegenüber gegenwärtigen Verfahren. Auch in der biomedizi- nischen und molekularbiologischen Forschung bedeutet daher die zelluläre Aufnahmer "nackter" Plasmid-DNA eine deutliche und nachhaltige Verbesserung des gegenwärtigen Standes der Technik.The cellular uptake of recombinant nucleic acids via the nucleotide sequences according to the invention, which are part of the nucleic acids themselves to be introduced, means a massive simplification compared to current methods, both in technical and biological terms. In biomedical and molecular biological research, too, the cellular uptake of "naked" plasmid DNA means a clear and sustainable improvement of the current state of the art.
Die Figuren zeigen:The figures show:
Fig. 1 ist eine schematische Darstellung eines erfindungsgemäßen Selektionsprotokolls zur Identifizierung von zellulären DNA-Importsignalen.1 is a schematic representation of a selection protocol according to the invention for the identification of cellular DNA import signals.
Fig. 2 zeigt in Balkendiagrammen die Effektivität der Aufnahme von selektierter Pool-DNA durch (A) ECV-304-Zellen bzw. (B) durch2 shows in bar graphs the effectiveness of the uptake of selected pool DNA by (A) ECV-304 cells or (B) by
BHK-21 -Zellen. Angegeben sind jeweils die intemalisierten DNA- Kopien bezogen auf eine Zelle. Die Fehlerbalken geben die Abweichung zweier Experimente wieder.BHK-21 cells. The internalized DNA copies are given in relation to one cell. The error bars show the deviation of two experiments.
Fig. 3 ist ein Balkendiagramm, das beispielhaft die zelluläre Aufnahme einzelner spezifischer DNA-Spezies (aus Pool 4) zeigt, deren Mittelwert bei 48 Kopien pro Zelle liegt. Der Mittelwert der Spezies des Ausgangs-Pools ist 7 Kopien pro Zelle.
Fig. 4 zeigt die Sequenzen der sieben Spezies der Fig. 3 mit den bestenFIG. 3 is a bar diagram which shows, by way of example, the cellular uptake of individual specific DNA species (from pool 4), the mean value of which is 48 copies per cell. The mean of the parent pool species is 7 copies per cell. Figure 4 shows the sequences of the seven species of Figure 3 with the best
Aufnahmeeigenschaften (Klone 14, 39, 40, 59, 61 , 113 und 131 ). Die fett markierten Sequenzen an den 5'- und 3'-Enden der Klone sind jeweils die zur Amplifikation verwendeten Primersequenzen.Uptake characteristics (clones 14, 39, 40, 59, 61, 113 and 131). The sequences marked in bold at the 5 'and 3' ends of the clones are the primer sequences used for amplification.
Das folgende Beispiel erläutert die vorliegende Erfindung näher, ohne sie einzuschränken.The following example explains the present invention in more detail without restricting it.
BEISPIELEXAMPLE
Selektionsverfahren zur Identifizierung von DNA-TransportsignalsequenzenSelection procedure for the identification of DNA transport signal sequences
In der Figur 1 ist das Selektionsprotokoll zur Identifizierung von DNA-Signalse- quenzen, welche die Aufnahme von DNA in Kulturzellen bewirken, gezeigt. Es wurden jeweils 10 μg eines DNA-Pools mit Zellen der Säugerzellinien BHK-21 bzw. ECV-304 in Kontakt gebracht. Die Länge der DNA-Spezies betrug 146 Basenpaare, umfassend einen randomisierten Anteil mit 100 Basenpaaren und terminale Primersequenzen mit je 23 Basenpaaren. Die Komplexität des DNA- Pools betrug 1013 unterschiedliche DNA-Moleküle bzw. Spezies. Die BHK-21 bzw. ECV-304-Zellen (jeweils etwa 107 Zellen) wurden mit dem DNA-Pool für 24 Stunden inkubiert. Nach dem ausgiebigen Waschen folgte die Isolierung der nukleinsäurehaltigen Bestandteile der Zellen (intrazelluläre Fraktion). Dieses Isolat diente sodann als Matrize für eine präparative PCR mit Hilfe von Primern, die an die terminalen Primersequenzen der eingesetzen DNA-Spezies bindeten. Im nächsten Selektionsschritt wurden die mit Hilfe der präparativen PCR erhaltenen und danach isolierten Amplifikate wieder mit den jeweiligen Zellen inkubiert. Wie die Fig. 2A und 2B zeigen, erfolgt mit Hilfe des in der Fig. 1 schematisch dargestellten Identifizierungsverfahrens nach drei Selektionszyklen bei ECV-304- Zellen eine deutliche Anreicherung von DNA-Spezies mit einem Faktor von fast sechs (Fig. 2A), während im Fall von BHK-21 -Zellen nach vier Selektionszyklen sogar eine 20fache Anreicherung im Vergleich zum Ausgangswert (Zyklus 0) erhalten wird (Fig. 2B). Wird nach diesem Selektionsschema der Punkt erreicht, an dem auch nach weiteren Selektrionsrunden keine weitere Verbesserung der
12 zellulären DNA-Aufnahme erfolgt, werden die selektierten DNA-Spezies kloniert und strukturell sowie funktionell charakterisiert.FIG. 1 shows the selection protocol for identifying DNA signal sequences which bring about the uptake of DNA in culture cells. In each case 10 μg of a DNA pool were brought into contact with cells from the BHK-21 or ECV-304 mammalian cell lines. The length of the DNA species was 146 base pairs, comprising a randomized portion with 100 base pairs and terminal primer sequences with 23 base pairs each. The complexity of the DNA pool was 10 13 different DNA molecules or species. The BHK-21 and ECV-304 cells (each about 10 7 cells) were incubated with the DNA pool for 24 hours. After extensive washing, the nucleic acid-containing components of the cells were isolated (intracellular fraction). This isolate then served as a template for a preparative PCR using primers that bound to the terminal primer sequences of the DNA species used. In the next selection step, the amplificates obtained with the help of the preparative PCR and then isolated were incubated again with the respective cells. As shown in FIGS. 2A and 2B, with the aid of the identification method shown schematically in FIG. 1, after three selection cycles in ECV-304 cells, DNA species are significantly enriched by a factor of almost six (FIG. 2A), while in the case of BHK-21 cells, even a 20-fold enrichment is obtained after four selection cycles compared to the starting value (cycle 0) (FIG. 2B). If, according to this selection scheme, the point is reached at which there is no further improvement in the results even after further selection rounds 12 cellular DNA uptake takes place, the selected DNA species are cloned and characterized structurally and functionally.
Zelluläre Aufnahme von mit dem Selektionsverfahren identifizierten DNA- SpeziesCellular uptake of DNA species identified by the selection method
Mit Hilfe des erfindungsgemäßen Selektionsverfahrens wurden DNA-Spezies identifiziert, die selektiv von Kulturzellen aufgenommen werden. In der Fig. 3 sind die Anreicherungen einzelner DNA-Spezies im Vergleich zum Ausgangspool (7 Kopien pro Zelle) in einem Balkendiagramm widergegeben. Der Mittelwert der gezeigten Spezies beträgt 48 Kopien pro Zelle. Den Wert des Ausgangspools übertreffen die sieben am stärksten angereicherten Spezies, deren Nukleotidsequenzen in der Fig. 4 gezeigt sind, um mindestens das Achtfache, während die DNA-Spezies mit der größten Anreicherung (Klon 113) darüber hinaus eine mehr als 36fache Anreicherung gegenüber dem Ausgangspool aufweist.
With the help of the selection method according to the invention, DNA species were identified which are selectively taken up by culture cells. 3 shows the enrichments of individual DNA species compared to the starting pool (7 copies per cell) in a bar chart. The mean of the species shown is 48 copies per cell. The value of the parent pool exceeds the seven most enriched species, the nucleotide sequences of which are shown in Fig. 4, by at least eight times, while the DNA species with the greatest concentration (clone 113) also more than 36 times the concentration compared to the parent pool having.
AnsprücheExpectations
1. Nukleinsäure, enthaltend eine erste Nukleotidsequenz mit mindestens einer Signalsequenz, welche die Aufnahme der Nukleinsäure in eine Zelle bewirkt.1. Nucleic acid containing a first nucleotide sequence with at least one signal sequence which causes the nucleic acid to be taken up into a cell.
2. Nukleinsäure nach Anspruch 1 , wobei die Nukleotidsequenz weiter minde- stens eine zu transportierende zweite Nukleotidsequenz und/oder eine oder mehrere mit der ersten und/oder zweiten Nukleotidsequenz kovalent verknüpfte und/oder komplexierte Komponenten enthält, die biologisch aktiv und keine Nukleinsäuren sind.2. The nucleic acid according to claim 1, wherein the nucleotide sequence further contains at least one second nucleotide sequence to be transported and / or one or more components covalently linked and / or complexed with the first and / or second nucleotide sequence, which are biologically active and are not nucleic acids.
3. Nukleinsäure nach Anspruch 2, wobei die zu transportierende zweite Nukleotidsequenz einen für mindestens ein Polypeptid und/oder für mindestens eine Antisense-Nukleinsäure und/oder für mindestens ein Ribozym kodierenden Bereich und/oder einen oder mehrere Kontrollbereiche umfaßt.3. Nucleic acid according to claim 2, wherein the second nucleotide sequence to be transported comprises a region coding for at least one polypeptide and / or for at least one antisense nucleic acid and / or for at least one ribozyme and / or one or more control regions.
4. Nukleinsäure nach Anspruch 3, wobei der (die) Kontrollbereich(e) einen Promotor und/oder einen Enhancer und/oder einen Operator und/oder ein Transkriptions/Translationsstartsignal und/oder ein Polyadenylierungssignal umfaßt (umfassen).4. The nucleic acid according to claim 3, wherein the control area (s) comprises a promoter and / or an enhancer and / or an operator and / or a transcription / translation start signal and / or a polyadenylation signal.
5. Nukleinsäure nach Anspruch 3 oder 4, wobei der (die) Kontrollbereich(e) zur Regulation der Expression des mindestens eine für mindestens ein Polypeptid und/oder für mindestens eine Antisense-Nukleinsäure und/oder für mindestens ein Ribozym kodierenden Bereichs befähigt ist (sind).
5. The nucleic acid according to claim 3 or 4, wherein the control region (s) is capable of regulating the expression of the at least one region coding for at least one polypeptide and / or for at least one antisense nucleic acid and / or for at least one ribozyme ( are).
Claims
14 14
6. Nukleinsäure nach einem der Ansprüche 1 bis 5, wobei die Zelle eine Säugerzelle ist.6. Nucleic acid according to one of claims 1 to 5, wherein the cell is a mammalian cell.
7. Vektor, der mindestens die Nukleinsäure nach einem der Ansprüche 1 bis 6 enthält.7. Vector containing at least the nucleic acid according to one of claims 1 to 6.
8. Wirtsorganismus, der mindestens die Nukleinsäure nach einem der Ansprüche 1 bis 6 oder den Vektor nach Anspruch 7 enthält.8. Host organism which contains at least the nucleic acid according to one of claims 1 to 6 or the vector according to claim 7.
9. Verfahren zur Herstellung der Nukleinsäure nach einem der Ansprüche 1 bis 6 oder des Vektors nach Anspruch 7, umfassend die Schritte:9. A method for producing the nucleic acid according to one of claims 1 to 6 or the vector according to claim 7, comprising the steps:
(a) Züchten des Wirtsorganismus nach Anspruch 8 in einem geeigneten Medium unter geeigneten Bedingungen und(a) culturing the host organism according to claim 8 in a suitable medium under suitable conditions and
(b) Isolieren des gewünschten Produkts aus dem Medium und/oder dem Wirtsorganismus.(b) isolating the desired product from the medium and / or the host organism.
10. Verfahren zur Identifizierung einer Nukleotidsequenz, die mindestens eine die Aufnahme einer Nukleinsäure in eine Zelle bewirkende Signalsequenz enthält, umfassend die Schritte: (a) Isolieren einer Nukleinsäure, die von Zellen selektiv aus einer Vielzahl von Nukleinsäuren aufgenommen wird, wobei die Vielzahl der Nukleinsäuren Nukleotidsequenzen aufweisen, die sich mindestens teilweise voneinander unterscheiden, und (b) Bestimmen der Nukleotidsequenz der in Schritt (a) isolierten Nu- kleinsäure.10. A method for identifying a nucleotide sequence which contains at least one signal sequence which causes a nucleic acid to be taken up into a cell, comprising the steps: (a) isolating a nucleic acid which is selectively taken up by cells from a large number of nucleic acids, the large number of nucleic acids Have nucleotide sequences that differ at least partially from one another, and (b) determining the nucleotide sequence of the nucleic acid isolated in step (a).
11. Verfahren nach Anspruch 10, wobei das Isolieren der Nukleinsäure11. The method of claim 10, wherein isolating the nucleic acid
(i) das Inkontaktbringen der Zellen für einen geeigneten Zeitraum mit der Vielzahl von Nukleinsäuren, wobei die Zellen selektiv Nukleinsäuren aus der Vielzahl von Nukleinsäuren aufnehmen,(i) contacting the cells with the plurality of nucleic acids for a suitable period of time, the cells selectively taking up nucleic acids from the plurality of nucleic acids,
(ii) das Isolieren der Nukleinsäure-haltigen Bestandteile der Zellen, (iii) das Detektieren der von den Zellen selektiv aus der Vielzahl der Nukleinsäuren aufgenommenen Nukleinsäuren in den Nukleinsäure- haltigen Bestandteilen der Zellen von (ii), und
(iv) das Isolieren der im Schritt (iii) detektierten, selektiv von den Zellen aufgenommenen Nukleinsäuren umfaßt.(ii) isolating the nucleic acid-containing components of the cells, (iii) detecting the nucleic acids selectively taken up by the cells from the large number of nucleic acids in the nucleic acid-containing components of the cells of (ii), and (iv) isolating the nucleic acids detected in step (iii) and selectively taken up by the cells.
12. Verfahren nach Anspruch 11 , das weiter12. The method of claim 11, the further
(v) das Wiederholen der Schritte (i) bis (iv), bis sich der Anteil der aus der Vielzahl von Nukleinsäuren selektiv aufgenommenen Nukleinsäuren an den Nukleinsäure-haltigen Bestandteilen der Zellen nicht mehr ändert, wobei jeweils im Schritt (i) die im Schritt (iv) isolierten Nukleinsäuren mit den Zellen in Kontakt gebracht werden, umfaßt.(v) repeating steps (i) to (iv) until the proportion of the nucleic acids selectively taken up from the large number of nucleic acids in the nucleic acid-containing constituents of the cells no longer changes, in each case in step (i) that in step (iv) isolated nucleic acids are contacted with the cells.
13. Verfahren nach Anspruch 11 oder 12, wobei das Detektieren gleichzeitig die Amplifikation der Nukleinsäuren umfaßt.13. The method according to claim 11 or 12, wherein the detection simultaneously comprises the amplification of the nucleic acids.
14. Verfahren nach einem der Ansprüche 11 bis 13, wobei das Detektieren eine PCR umfaßt.14. The method according to any one of claims 11 to 13, wherein the detection comprises a PCR.
15. Verfahren nach einem der Ansprüche 11 bis 14, das weiter ein oder mehrere Waschschritte umfaßt.15. The method according to any one of claims 11 to 14, further comprising one or more washing steps.
16. Kit zur Einschleusung von Nukleinsäuren in Zellen, umfassend die Nukleinsäure nach einem der Ansprüche 1 bis 6 und/oder den Vektor nach Anspruch 7 und/oder den Wirtsorganismus nach Anspruch 8.16. Kit for introducing nucleic acids into cells, comprising the nucleic acid according to one of claims 1 to 6 and / or the vector according to claim 7 and / or the host organism according to claim 8.
17. Arzneimittel, enthaltend die Nukleinsäure nach einem der Ansprüche 1 bis 6 oder den Vektor nach Anspruch 7 und gegebenenfalls einen pharmazeutisch verträglichen Träger und/oder Hilfsstoff und/oder ein Verdünnungsmittel.17. Medicament containing the nucleic acid according to one of claims 1 to 6 or the vector according to claim 7 and optionally a pharmaceutically acceptable carrier and / or auxiliary and / or a diluent.
18. Verwendung der Nukleinsäure nach einem der Ansprüche 1 bis 6 oder des Vektors nach Anspruch 7 oder des Wirtsorganismus' nach Anspruch 8 zur18. Use of the nucleic acid according to one of claims 1 to 6 or of the vector according to claim 7 or of the host organism according to claim 8 for
Herstellung eines gentherapeutischen Arzneimittels.
1/4Manufacture of a gene therapy drug. 1.4
Fig. 1Fig. 1
Pool-DNAPool DNA
• Länge der DNA-Spezies: 146 bp • Length of the DNA species: 146 bp
• Randomisierter Anteil: 100 bpRandomized portion: 100 bp
• Terminale Primersequenzen: 23 bp• Terminal primer sequences: 23 bp
• Komplexität: 1013 Spezies• Complexity: 10 13 species
• Menge: 10ug• Amount: 10ug
z.B: BHK, Helae.g .: BHK, Hela
Amplifikation mit Hilfe oder ECV 304 präparativer PCRAmplification with the help or ECV 304 preparative PCR
Inkubation mit Pool-DNAIncubation with pool DNA
Intrazelluläre Fraktion
Intracellular fraction
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU58230/00A AU5823000A (en) | 1999-07-16 | 2000-06-30 | Cellular absorption of dna |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE1999133506 DE19933506A1 (en) | 1999-07-16 | 1999-07-16 | Cellular uptake of DNA |
| DE19933506.0 | 1999-07-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2001005993A1 true WO2001005993A1 (en) | 2001-01-25 |
Family
ID=7915092
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP2000/006155 WO2001005993A1 (en) | 1999-07-16 | 2000-06-30 | Cellular absorption of dna |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU5823000A (en) |
| DE (1) | DE19933506A1 (en) |
| WO (1) | WO2001005993A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0388758A1 (en) * | 1989-03-16 | 1990-09-26 | BOEHRINGER INGELHEIM INTERNATIONAL GmbH | Protein-polycation conjugate |
| EP0890362A1 (en) * | 1997-07-01 | 1999-01-13 | Transgene S.A. | Compositions useful for transferring therapeutically active substances into a target cell, and their use in gene therapy |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5569754A (en) * | 1993-06-11 | 1996-10-29 | Board Of Regents, University Of Tx Systems | RNA import elements for transport into mitochondria |
| US5888727A (en) * | 1996-05-23 | 1999-03-30 | Wisconsin Alumni Research Foundation | Method of inhibition of nucleo-cytoplasmic transport by M protein of vesicular stomatitis virus |
-
1999
- 1999-07-16 DE DE1999133506 patent/DE19933506A1/en not_active Ceased
-
2000
- 2000-06-30 AU AU58230/00A patent/AU5823000A/en not_active Abandoned
- 2000-06-30 WO PCT/EP2000/006155 patent/WO2001005993A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0388758A1 (en) * | 1989-03-16 | 1990-09-26 | BOEHRINGER INGELHEIM INTERNATIONAL GmbH | Protein-polycation conjugate |
| EP0890362A1 (en) * | 1997-07-01 | 1999-01-13 | Transgene S.A. | Compositions useful for transferring therapeutically active substances into a target cell, and their use in gene therapy |
Non-Patent Citations (2)
| Title |
|---|
| NAKAMURA MASARU ET AL: "Uptake and gene expression of naked plasmid DNA in cultured brain microvessel endothelial cells.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 245, no. 1, 7 April 1998 (1998-04-07), pages 235 - 239, XP002149579, ISSN: 0006-291X * |
| TINLAND BRUNO ET AL: "Agrobacterium tumefaciens transfers single-stranded transferred DNA (T-DNA) into the plant cell nucleus.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 91, no. 17, 1994, 1994, pages 8000 - 8004, XP002149578, ISSN: 0027-8424 * |
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| DE19933506A1 (en) | 2001-01-25 |
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