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WO2001005993A1 - Absorption cellulaire d'adn - Google Patents

Absorption cellulaire d'adn Download PDF

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Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
cells
nucleic acids
dna
isolating
Prior art date
Application number
PCT/EP2000/006155
Other languages
German (de)
English (en)
Inventor
Georg Sczakiel
Maik Lehmann
Original Assignee
Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts filed Critical Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority to AU58230/00A priority Critical patent/AU5823000A/en
Publication of WO2001005993A1 publication Critical patent/WO2001005993A1/fr

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Classifications

    • 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

L'invention concerne un nouveau système d'absorption cellulaire d'acides nucléiques, de substances contenant des composants d'acide nucléique, ou d'informations génétiques. L'invention concerne notamment un acide nucléique contenant une séquence nucléotidique ayant au moins une séquence-signal qui provoque l'absorption de l'acide nucléique dans une cellule, leur procédé de production et un procédé d'identification de telles séquences nucléotiques.
PCT/EP2000/006155 1999-07-16 2000-06-30 Absorption cellulaire d'adn WO2001005993A1 (fr)

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 (de) 1999-07-16 1999-07-16 Zelluläre Aufnahme von DNA
DE19933506.0 1999-07-16

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WO2001005993A1 true WO2001005993A1 (fr) 2001-01-25

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AU (1) AU5823000A (fr)
DE (1) DE19933506A1 (fr)
WO (1) WO2001005993A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0388758A1 (fr) * 1989-03-16 1990-09-26 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Conjugé protéine-polycation
EP0890362A1 (fr) * 1997-07-01 1999-01-13 Transgene S.A. Compositions utiles pour le transfert des substances thérapeutiquement actives dans une cellule ciblé et leur utilisation dans la thérapie génique

Family Cites Families (2)

* Cited by examiner, † Cited by third party
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0388758A1 (fr) * 1989-03-16 1990-09-26 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Conjugé protéine-polycation
EP0890362A1 (fr) * 1997-07-01 1999-01-13 Transgene S.A. Compositions utiles pour le transfert des substances thérapeutiquement actives dans une cellule ciblé et leur utilisation dans la thérapie génique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
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 (de) 2001-01-25

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