EP1141364A1 - Ausnutzung zelleigener transportsysteme zum transfer von nukleinsäuren durch die kernhülle - Google Patents

Ausnutzung zelleigener transportsysteme zum transfer von nukleinsäuren durch die kernhülle

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Publication number
EP1141364A1
EP1141364A1 EP00903497A EP00903497A EP1141364A1 EP 1141364 A1 EP1141364 A1 EP 1141364A1 EP 00903497 A EP00903497 A EP 00903497A EP 00903497 A EP00903497 A EP 00903497A EP 1141364 A1 EP1141364 A1 EP 1141364A1
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EP
European Patent Office
Prior art keywords
dna
transport agent
module
agent according
nls
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP00903497A
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German (de)
English (en)
French (fr)
Inventor
Gregor Siebenkotten
Rainer Christine
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lonza Cologne GmbH
Original Assignee
Amaxa AG
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Filing date
Publication date
Priority claimed from DE19933939A external-priority patent/DE19933939A1/de
Application filed by Amaxa AG filed Critical Amaxa AG
Publication of EP1141364A1 publication Critical patent/EP1141364A1/de
Withdrawn legal-status Critical Current

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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C07K14/003—Peptide-nucleic acids (PNAs)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a core transport agent, a gene transfer system comprising the core transport agent, a method for transporting DNA into the nucleus of eukaryotic cells, using the core transport agent and the use of the core transport agent in gene therapy for the treatment of cancer, viral infections, diseases of the nervous system, transplant rejection, as well as monogenic or polygenic hereditary diseases.
  • the active transport into the nucleus is necessary for the transfer of genetic material in all cells that do not divide in the period up to the desired expression of the genetic material.
  • a core transport system for nucleic acids is very important because it enables the efficient transfer of DNA into cells that do not or only rarely divide (Dowty et al., 1995, Wilke et al., 1996). Most primary cells fall into this group. Primary cells are of the highest scientific interest because, on the one hand, these freshly isolated cells from an organism reflect the functional state of the cell type much better than cell lines derived from them, and on the other hand, they are target cells for gene therapies.
  • a core transport system also increases the efficiency of DNA transfer to established cell lines by allowing cells that have not divided in the period from the start of the transfer to analysis to express transferred genetic material.
  • the place of action of the genetic material is the cell nucleus.
  • the double membrane that surrounds the cell nucleus has pores. Small molecules can diffuse through these pores. Proteins that are larger than approx. 50 kDa require a nuclear localization signal (NLS), which must be recognized by a transport machine so that they can get into the nucleus. A sufficient signal typically consists of four to eight amino acids and is rich in positively charged amino acids arginine and lysine and has proline. It is evolutionarily highly conserved, so that NLS from mammalian proteins also work in yeast. Heterologous NLS can also be used as a tool to transport target molecules into the cell nucleus. For this purpose, NLS can be built into the sequence of cytoplasmic proteins at a relatively arbitrary point or chemically coupled as a peptide to proteins or even to gold particles (review article: Görlich, 1998).
  • HIV and other lentiviruses that are able to infect resting cells use viral proteins and the cell's own transport machinery to transfer their DNA to the nucleus.
  • the NLS in the Vpr and in the matrix protein of the HIV pre-integration complex are essential for the infection of cells that do not divide (Naldini et. Al., 1996). Although little is known about how viruses bring their genome into the nucleus, the help of viral structural proteins with NLS could be a general principle. The following observations also support this.
  • a specific mutation in the HSV capsid protein prevents the transport of viral DNA into the nucleus (Batterson et al., 1983).
  • Adenovirus DNA is transported into the nucleus together with the hexon protein of the dissolved capsid (Greber et al., 1993).
  • SV40 DNA is transported into the cell nucleus by a viral protein (most likely Vp3) that remains associated with the DNA (Nakanishi et al., 1996).
  • Vp3 viral protein
  • Two bacterial proteins with NLS are responsible for the import of T-DNA from Agrobacterium tumefaciens into plant cell nuclei (Citovsky et al., 1994).
  • mutant variants of, e.g. B., HIV, adenovirus and herpes virus used as a DNA transfer vehicle for the development of gene therapy approaches.
  • helper cell lines are used in such systems in which the release of less strongly mutated virus genomes is not excluded.
  • the handling of these systems is complex.
  • Kaneda et. al. (1989) and Dzau and Kaneda, (1997, Patent EP or US 5,631,237) describe a gene transfer system which is based on the use of Sendai viruses, liposomes and added proteins which are intended to support the nuclear transport of the DNA.
  • the working group uses HMG-1 ("high mobility group 1 protein"), a basic non-histone protein of chromatin, which binds to DNA.
  • HMG-1 binds DNA over a long basic range. It is localized in the core, but has no known NLS.
  • HMG-1 protein forms complexes in vitro with vector DNA. The production of cleaned HMG-1 is complex.
  • HMG-1 is used here as a DNA-complexing transfection reagent for the passage of DNA through the cell membrane. The efficiency is low.
  • Wako BioProducts (Richmond, VA, USA) (1997) offered the proteins HMG-1 and -2 to mediate nuclear transport as an additive to lipofection reagents.
  • Gopal (US Pat. No. 5,670,347) describes a peptide which consists of a DNA-binding basic region, a flexible “hinge” region and an NLS. Since the DNA binding is also achieved here via positive charges of the amino acids, the reagent forms with the DNA complexes, which are also supposed to serve for the transport through the cell membrane. It is not clear why the NLS sequence should not be involved in the DNA binding, so the actual signal for the nuclear transport proteins is probably again masked by the DNA, how the DNA is linked to the peptide.
  • Gerhard et al. (DE-OS 195 41 679) propose NLS-polylysine conjugates for gene transfer.
  • the resulting complexes of cationic polylysine, cationic NLS and DNA mask the actual nuclear transport signal as long as it is connected to the DNA.
  • Szoka (PCT from 1993 Claim 23-27) couples NLS peptides to DNA via an intercalating agent. After a pre-incubation of vector and peptide (in a ratio of 1: 300), the
  • the SV40 peptide used is able to complex DNA due to its strong positive charge. Complexation of DNA with cationic peptides leads to an increase in the efficiency of lipofection by improving the efficiency of the passage through the cell membrane (Sorgi et al., 1997, cf. Hawley-Nelson et. Al., 1997). Kerat transport is at least hampered by this, at least when large complexes are formed (see above). Since the NLS peptides used themselves due to their charge to the
  • NLS peptide binds itself to the DNA as before and complexes it.
  • the problem of a direct binding of the NLS peptide to the DNA is not dealt with.
  • Hawley-Nelson et al. (US Patent 5736392) describe a comparable system.
  • An NLS peptide is mixed with vector DNA either directly or after covalent coupling to a DNA-binding molecule.
  • the resulting complexes are then used for lipofection (or other transfections).
  • the addition of a polycationic peptide without NLS increases the transfection efficiency even more than the addition of a cationic NLS.
  • the coupling of spermidine to the NLS peptide does not further increase the transfection efficiency. So here too the amplification effect can be explained solely by the complexation of the DNA via cationic peptides. Since that The presence of NLS does not further increase the transfection efficiency, it can be assumed that the recognition sequence for the core transport machinery is also masked here.
  • the company TIB Molbiol (brochure 1998) describes the transport of PNA oligonucleotides with a C-terminal NLS (nuclear localization signal) peptide in order to specifically suppress the gene expression of selected genes.
  • the NLS is used here to transport the PNA oligonucleotides into the nucleus so that they can hybridize there with the target sequence.
  • a core transport agent consisting of two modules A and B, module A specifically binding to DNA and not by unspecific binding leading to the formation of complexes with more than one DNA molecule and module B a nuclear localization signal or a non-NLS Contains signal that does not bind non-specifically to DNA.
  • a preferred core transport agent according to the invention comprises a module A which binds sequence-specifically to the DNA and / or binds specifically to DNA ends.
  • a core transport agent in which module A is a synthetic peptide, a protein or a PNA (peptide nucleic acid) is particularly preferred.
  • module B contains an expanded nuclear localization signal which, owing to its charges, does not form any complexes with DNA.
  • a core transport agent in which module B contains an expanded nuclear localization signal which has an approximately neutral total charge is preferred.
  • a core transport agent in which module B contains an expanded nuclear localization signal which comprises a nuclear localization signal and flanking negatively charged amino acids is particularly preferred.
  • An NLS sequence does not have to be identical to a natural one occurring NLS sequence, it can also be an amino acid sequence based on theoretical considerations, as long as it functions as an NLS.
  • Module B may further contain peptide sequences or non-peptide components that are not to be counted directly to the nuclear localization signal or extended nuclear localization signal. Preferably a component that increases the distance between the core localization signal and module A.
  • the invention further relates to a gene transfer system comprising a core transport agent according to the invention and a cationic lipid, peptide, polyamine or cationic polymer.
  • the invention relates to a method for the transport of DNA into the nucleus of eukaryotic cells, preferably primary cells, the cells being transfected with the DNA to be transported and the core transport agent according to the invention according to the prior art methods.
  • An additional embodiment relates to the use of the core transport agent according to the invention in gene therapy, in particular for the treatment of cancer, viral infections, diseases of the nervous system, transplant rejection and monogenic or polygenic hereditary diseases.
  • unspecific binding of the key localization signal to DNA means a binding which has the consequence that the nuclear localization signal is no longer completely recognizable by the nuclear transport machinery.
  • module A means on the one hand a sequence-specific binding, in which the sequence of the DNA nucleotides is decisive for the binding, on the other hand a covalent binding to DNA by DNA single or double stranded ends is conveyed.
  • extended core localization signal means that a core localization signal additional, flanking Has amino acids.
  • An extended nuclear localization signal is preferred which has 2-40, preferably 4-20, additional, flanking amino acids.
  • extended nuclear localization signal which due to its charges does not form a complex with DNA
  • module B contains a nuclear localization signal, the charge of which is distributed in such a way that it does not interact non-specifically with DNA and therefore remains fully accessible to the core transport machinery.
  • approximately neutral total charge means that the extended part of the core localization signal has negatively charged amino acids in order to balance the positive charge of the actual core localization signal, so that no more than three positive charges in excess occur in the entire area of the extended core localization signal.
  • the core transport agent according to the invention has the advantage that it does not lead to complexation of the DNA. Another advantage is that the core localization signal remains freely accessible to the core transport machinery. The avoidance of large DNA complexes which hinder nuclear transport and the absence of the nuclear localization signals for the nuclear transport machinery when using the nuclear transport agents according to the invention lead to a significantly more efficient transport of the DNA into the nucleus.
  • Module A binds specifically to DNA and does not lead to the formation of complexes with more than one DNA molecule.
  • Module A binds either sequence-specifically (ie not unspecifically due to positive charges alone) or covalently to DNA ends.
  • Module A can be a peptide of different lengths or a protein or a PNA sequence (Nielson et al., 1991) or another substance which binds to nucleic acids in a sequence-specific manner.
  • Module A can also be a recombinant protein that is sequence-specific to DNA binds such.
  • lac repressor or a high affinity mutant thereof (Kolkhof, 1992, Fieck et al., 1992) or a retroviral integrase that binds sequence-specifically to DNA ends (with an LTR core sequence) (Ellison and Brown, 1994) .
  • Covalent attachment to a strand end can be biological, e.g. through the topoisomerase I of the poxyvirus, if the strand end of a linear DNA carries a sequence which as a "suicide substrate" enables a topoisomerase cut but no religation (Shuman, 1994).
  • Module B is a nuclear localization signal or a non-NLS signal that does not bind non-specifically to DNA.
  • non-NLS signals means signals which are not nuclear localization signals, but which, in the context of transfection, gene therapy or DNA vaccination, serve to transport the DNA into the cell or to transport DNA within the cell.
  • Non-NLS signals include: ligands for cellular surface structures, DNA uptake, e.g. the receptor-mediated can mediate; Peptides that destabilize membranes, e.g. to promote the early release of DNA from the endosomes; Signals that mediate binding to transport structures in the cell in order to promote intracellular transport to the nucleus.
  • the nuclear localization signal is preferably an extended nuclear localization signal (as defined above) which, owing to its charges or spatial orientation in relation to the DNA-binding module A, does not form any complexes with DNA.
  • the nuclear localization signal can be produced synthetically or as part of a protein.
  • a nuclear localization signal as is used in the core transport agent according to the invention, those signal sequences - with and without flanking regions - are used which do not bind to the DNA via their positive charges in such a way that these charges, the one make up an essential part of most nuclear transport signals as masked signals for the nuclear transport machinery.
  • module B can contain peptide sequences or non-peptide components which are not attributable to the core localization signal or the extended core localization signal. They are preferably used to improve the steric alignment of the core localization signal, in particular to increase the distance from the DNA.
  • IgM immunoglobulin M
  • NLS is part of a sequence-specific protein that binds to the DNA, the risk of this sequence (s) being masked by the DNA is relatively low.
  • extended NLS sequences can also be used here because of the higher efficiency.
  • non-classical NLS such as an NLS from the influenza Vs "nucleoprotein” (Wang et al., 1997, Neumann et al., 1997) that does not have a large excess of positive charges or does not get into the nucleus via the classic transport route can be used.
  • a non-NLS signal is bound to existing vector sequences via PNA (as module A).
  • PNA as module A
  • the binding between PNA and vector is sequence specific. This makes it possible to couple such non-NLS signals to almost all common expression vectors without having to change them.
  • sequence-specific binding of PNA to the DNA only those DNA sequences of the vector are used whose masking by PNA does not significantly damage the later intended use of the DNA.
  • expression vectors mainly sequences in the plasmid backbone are used, especially those that are found in most common expression vectors (e.g. promoter of the ampicillin resistance gene), but binding in the non-coding strand in the expression area is also possible.
  • An advantage of sequence-specific binding is that simple and rapid binding of the PNA-peptide hybrid to the DNA takes place. In example 2, e.g. a simple and fast binding reaction (5 min., 65 ° C.) of PNA-peptide hybrids to double-stranded DNA was shown. A spacer can lie between the PNA portion and the actual signal.
  • the spacer can serve to increase the distance from signal to DNA, e.g. to reduce steric disabilities.
  • the spacer can also serve to introduce a predetermined breaking point, e.g. to allow the separation of a ligand in the endosomal milieu via which the DNA is bound to an endocytosed cell surface receptor.
  • the present invention makes resting and weakly dividing active cells transfectable to a percentage, which enables a subsequent analysis.
  • Most cells (primary cells) freshly isolated from the body of an animal or human do not divide or so rarely that DNA that has been successfully transported across the cell membrane is inactivated before it can get into the nucleus and be expressed. Until now, this has resulted in these primary cells being untransfectable unless they were artificially stimulated to divide in culture. However, this always has the consequence that they differ from remove original condition.
  • a technique for transfection of primary cells enables the analysis of genetic material under the original conditions of a body cell, which is of great importance for the research of the mode of action of genes and for the research of processes within a body cell.
  • the teaching according to the invention which makes primary cells transfectable, also represents an essential step towards a completely artificial gene transfer system for gene therapy.
  • a gene transfer system must have three functional components: a component for the passage of DNA through the cell membrane, for which cationic lipids and other cationic polymers have proven to be relatively well suited, another for the transfer of the DNA into the nucleus of the (mostly division-inactive) target cells and a third, which ensures the integration of the DNA into the genome.
  • an efficient agent that can serve as a second component is described here for the first time.
  • a completely artificial gene transfer vehicle that can be used in gene therapy is expected to be easier and cheaper to manufacture and easier to handle than the viral systems currently in use and is not subject to the inherent risks of these systems.
  • Gene therapy approaches are e.g. for the treatment of cancer, AIDS and various inherited diseases have been proposed and will play a very large role in medicine.
  • the nuclear transport agents described according to the invention increase the efficiency of the transfection into those culture cells which were previously transfectable, since those cells can now also incorporate DNA into the nucleus which do not divide in the time window between the passage of the DNA through the cell membrane and analysis. This is important because even with many established cell lines, increasing the transfection efficiency would make the analysis easier and would reduce costs due to the lower need for cell material. Of course, this also applies to all intermediate stages between primary cells and established cell lines.
  • NLS-PNA core transport reagents were used. Such PNA sequences were used which are able to migrate into the DNA double strand (Nielson et al., 1991, Nielson, US Pat. No. 5,539,082).
  • the peptides used contain as peptide components: either 1) "SV21": NH 2 -GKPTADDQHSTPPKKKRKVED-COOH (peptide 1; SEQ ID NO: 1), or
  • the cuvette was incubated for a further 10 min at 37 ° C. before the cells were plated out in preheated medium.
  • pMACS4.1 an expression vector for human CD4
  • the cells were transfected with pMACS4.1 (an expression vector for human CD4) using the method described and the cell division was measured as follows.
  • CFDA carboxyfluorescein diacetate succinimide ester
  • the flow cytometer (FACSCalibur) was then used to determine at the single cell level that cells which had not divided (full green fluorescence) expressed the transfected gene (dark red fluorescence after staining with Cy5-coupled anti-CD4 antibody).
  • PNA-peptide hybrids were bound to double-stranded DNA.
  • An existing vector sequence can be labeled almost quantitatively (> 90%) via PNA with an NLS peptide within 5 min.
  • For the binding of heat-labile components via PNA an incubation at 37 ° C for one hour is sufficient to label most of the DNA (Table 1).
  • 100 ng of an expression vector were incubated in TE (pH 7.8) with 25 ⁇ M different PNA-peptide hybrids at 65 ° C. or 37 ° C. for five minutes to three hours.
  • the PNA sequence NH2-CTCTTCCTTTTTC-COOH (SEQ ID NO: 6) used binds in the promoter region of the ampicillin resistance gene.
  • the binding mixtures were then implemented with the restriction endonuclease Earl.
  • the cleaved DNA was stained with YOYO (Molecular Probes, Inc., Eugene, OR, USA), separated on an agarose gel and with a fluorescence reader (Image Plate Reader FLA 2000, evaluation software L-Process, version 1.6, Fuji Photo Film Co ., Ltd., Tokyo).
  • YOYO Molecular Probes, Inc., Eugene, OR, USA
  • the binding reaction of the PNA to the DNA is simple, robust and fast.
  • the binding is practically irreversible and therefore suitable for cellular transport processes.
  • Peptides and PNA are cheaper to manufacture and store longer and easier than proteins.
  • active nuclear transport of transfected DNA means that it can be expressed earlier than DNA that is not transported. Assuming that transfected DNA persists in the cytoplasm long enough, the expression rates of transfected DNA with and without a nuclear transport reagent should gradually approach each other in strongly dividing cells. This is due to the fact that transfected DNA present in the cytoplasm can get into the nucleus during cell division. With aphidicolin, the division activity and thus also the transfectability can be greatly reduced, active nuclear transport cancels this effect of the reduced transfection efficiency.
  • a high-affinity binding mutant of the lac repressor from E. coli was used as the sequence-specific DNA-binding protein. This mutant has a binding constant of 10 "15 M for the lac operator sequence (Kolkhof, 1992). The high affinity is achieved by an amino acid exchange serine 61 to leucine.
  • the nuclear transport proteins used here have a deletion of the last thirty C-terminal amino acids (positions 331-360) and an exchange of the leucine in position 330 to a serine. These mutant proteins form homodimers instead of homotetramers and therefore have only one DNA binding site instead of two. However, tetramers can also be used for a core transport agent according to the invention.
  • the dimer variants were extended by one NLS each at the N-terminus:
  • NLS 1 MPKKKRKV-MKP VTLYDVA
  • N2D NLS2 MEEDTPPKKKRKVEDL-KPVTLYDVA
  • NLS1 corresponds to the NLS of the SV40 Vim's large T antigen.
  • NLS2 is a total charge neutral hybrid of the SV40-NLS and the N-terminal flanking region of the NLS of the polyoma virus VP2 protein.
  • Lac operator sequences occur in a number of expression vectors and can easily be linked to any sequence as an overhang of a PCR primer.
  • lac operator sequences were used for binding studies: the naturally occurring operator: AATTGTGAGC GGATAACAATT and a perfectly palindromic operator sequence: AATTGTGAGC GCTCACAATT.
  • 0.7 ng of a radioactively labeled DNA fragment of 1 kb in length was broken down into fragments of 914 and 86 bp in length by restriction digestion and incubated with lac represor NLS-1 dimer or NLS-2 dimer for 30 min at room temperature .
  • the fragments were then separated on a polyacrylamide gel (FIG. 2).
  • the 86 bp fragment which contains a lac operator, is retarded by specific binding. Nonspecific binding leads to the retardation of the 914 bp fragment without a lac operator. With complete specific binding, almost no non-specific binding can be determined.
  • the binding takes place and is stable under very different conditions, e.g. in the RPMI cell culture medium, in 150 mM sodium chloride or a buffer of 10 mM Tris / HCl (pH 7.2), 10 mM potassium chloride and 3 mM magnesium acetate, with both operator sequences tested.
  • lac repressor NLS mutants were treated with 2 ⁇ g (100 pmol) of a double-stranded DNA of 30 bp length, which was labeled with the fluorescent molecule Cy5 at both ends, in 300 ⁇ l 10 mM Tris / Cl ( pH 7.2)), 10 mM KC1, 3 mM Mg acetate and 50 ⁇ g / ml BSA for 30 min at room temperature.
  • a mixture of DNA bound to lac repressor mutants and fluorescein-labeled BSA (BSA-FITC) was microinjected into the cytoplasm in 50 NIH3T3 cells in each case (Eppendorf Transjector 5246 with femtotips, 0.5 ⁇ m diameter, injection pressure 55 hPa, Injection time 0.5 sec).
  • a fluorescence microscopic evaluation was carried out 10 to 15 min after the injection (see FIG. 3). If the cytoplasmic injection is successful, (BSA-FITC) is only in the cytoplasm (la, 2a, 3a).
  • Lac-Repressor-NLS can increase transfection efficiency by a factor of 3-4 4 h after transfection.
  • adherent NIH3T3 were cultivated to confluence before the transfection, which leads to a major inhibition of division. 4 h after the transfection, very few cells have divided.
  • the period in which the already low division activity is relevant for the transfection in this example is further shortened by the fact that the transfected, endocytotically recorded DNA first has to leave the endosomes and then the complex with the cationic transfection reagent before it can enter the core can be transported and expressed.
  • Lipofectamine-DNA complexes probably last much longer than complexes with polyethylene imine, which means that the effect of lac repressor NLS with Lipofectamine is less pronounced.
  • lac repressor NLS with Lipofectamine is less pronounced.

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EP00903497A 1999-01-08 2000-01-03 Ausnutzung zelleigener transportsysteme zum transfer von nukleinsäuren durch die kernhülle Withdrawn EP1141364A1 (de)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE19900513 1999-01-08
DE19900513 1999-01-08
DE19933939A DE19933939A1 (de) 1999-01-08 1999-07-20 Ausnutzung zelleigener Transportsysteme zum Transfer von Nukleinsäuren durch die Kernhülle
DE19933939 1999-07-20
PCT/DE2000/000061 WO2000040742A1 (de) 1999-01-08 2000-01-03 Ausnutzung zelleigener transportsysteme zum transfer von nukleinsäuren durch die kernhülle

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CN1262668C (zh) 2006-07-05
US6521456B1 (en) 2003-02-18
IL143983A0 (en) 2002-04-21
JP3997056B2 (ja) 2007-10-24
AU764660B2 (en) 2003-08-28
WO2000040742A1 (de) 2000-07-13
CA2358040C (en) 2008-06-10
JP2002534437A (ja) 2002-10-15
AU2533700A (en) 2000-07-24
CA2358040A1 (en) 2000-07-13
JP2006345867A (ja) 2006-12-28
CN1339068A (zh) 2002-03-06

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