EP0900281A1

EP0900281A1 - Composition for the transfection of higher eucaryotic cells

Info

Publication number: EP0900281A1
Application number: EP97904426A
Authority: EP
Inventors: Ernst Wagner; Karl Mechtler; Antoine Kichler
Original assignee: Boehringer Ingelheim International GmbH
Current assignee: Boehringer Ingelheim International GmbH
Priority date: 1996-02-15
Filing date: 1997-02-13
Publication date: 1999-03-10
Also published as: WO1997030170A1; CA2246227A1; MX9805507A; DE19605548A1; JP2000504579A

Abstract

The invention concerns a composition for the transfection of higher eucaryotic cells. A complex of a nucleic acid to be expressed in the cell and a cationic lipid present in a suboptimal concentration for transfection contains one or a plurality of membrane-active acid peptides and optionally helper lipid(s). The ratio of the total number of positive charges to the total number of negative charges in the composition is between approximately 0 and approximately 3.

Description

Composition for transfection of higher eukaryotic cells

The invention relates to the transfection of higher eukaryotic cells, in particular mammalian cells.

There is a need for an efficient system for the introduction of nucleic acid into living cells, especially in the context of gene therapy. Genes are introduced into cells to achieve the synthesis of therapeutically effective gene products in vivo.

The most advanced and widespread technologies for the use of nucleic acids in gene therapy for the transfer of genes into the cell have been based on viruses, particularly retroviruses and adenoviruses (Miller, 1992; Mulligan, 1993; Berkner; 1988) ), and cationic lipids (Behr, 1994). Alternative strategies for gene transfer are based on mechanisms that the cell uses to transport macromolecules. An example of this is the import of genes into the cell via the pathway of receptor-mediated endocytosis (e.g. Wu and Wu, 1987, Wagner et al., 1990, EP-AI 0388 758).

Of the synthetic gene transfer vehicles developed over the past decade, mono- and polycationic amphipathic lipids that can complex and condense DNA have proven particularly promising (Behr, et al., 1989; Feigner, et al., 1987 and 1994; Leventis, et al., 1990; Gao, et al., 1991; Rose, et al., 1991; Hawley-Nelson, et al., 1993; Weibel, et al., 1995; Solodin, et al., 1995). Efforts to increase the performance of gene transfer methods based on cationic lipids have hitherto focused on the direct modification of the cationic lipid, for example by means of acyl groups, spacer arms or hydrophilic sections (Remy, et al., 1994; Feigner, et al., 1994 ). Despite improved results due to such measures, much is still unclear about the entry mechanism of lipid / DNA particles into the cell. Based on recent publications (Zabner, et al., 1995), the main transport route for these particles is thought to be via endocytosis.

In vivo, cationic lipids show the best transfection results with a clearly positive excess charge. For example, for the lipospermin DOGS (dioctadecylamidoglyclyspermin, commercially available under the trade name "Transfectam") a three to six-fold excess of positive charges in relation to the DNA than for the

Transfection efficiency proven optimal (Barthel, et al., 1993). The positive charges promote the binding of the complex and its absorption into the cells. In addition, Transfectam has a buffering effect on the endosomes (the pKa value of the least basic secondary amine of lipospermin, which is decisive for the buffering effect, is approximately 5.4; Behr, 1994) and therefore protects the DNA on the one hand against enzymatic degradation and on the other hand causes it there is an osmotic swelling and subsequent destabilization of the buffered endosomes. A strongly positive charge of the lipid / DNA complexes thus has the disadvantage, inter alia, that the lipid / DNA particles only have a very short half-life when used in vivo. In order to improve the efficiency of the lipopolyamines DOGS and DPPES (palmitoylphosphatidylethanolamine) with a small positive charge excess (ratio positive: negative charges ≤. 2, preferably 0.5 to 1.5), the addition of adjuvants which can associate with the lipopolyamine / nucleic acid complex has been proposed , for example the addition of so-called "helper lipids".

By Kamata, et al. , 1994, was the efficiency of gene transfer with Lipofectin, a 1: 1 mixture of the monocationic lipid DOTMA (N- [l- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride) and the helper lipid DOPE (dioleylphosphatidylethanolamine), with a positive charge excess of 1.25, was increased by 3 to 5-fold using peptides.

Similarly, it was proposed in WO 95/02698 to use cationic lipids in combination with membrane-active peptides of enveloped viruses, for example for the transfection of cells. of influenza viruses, using one of the peptides described by Kamata et.al., 1994 or a peptide derived from the glycoprotein of Vesicular Stomatitis Virus in combination with lipofectamine, a 3: 1 mixture of the polycationic lipid 2,3-dioleyloxy-N- [ 2 (spermincarboxamido) ethyl] - N, N-dimethyl-l-propanaminium trifluoroacetate (DOSPA) and DOPE were used and with an optimally determined positive charge excess of approx. 20, the transfection efficiency was improved by up to 12 times.

On the other hand, cationic lipids conjugated with viral peptides have given them the fusogenic or karyophilic properties of viruses confer disappointing results at optimal lipid concentration (Remy et al., 1995).

The object of the present invention was to provide a new gene transfer system based on cationic lipids.

When solving the task, the question was first asked whether it is the steps following endocytosis, such as the transport of DNA from the endosome into the cytoplasm and further into the nucleus, which represent the bottleneck for successful gene transfer with cationic lipids.

It was found that the addition of membrane-active influenza apeptides causes only a slight increase in gene expression (1.5 to 5-fold) with transfectam when this is used at optimal concentrations, ie with a high positive charge excess. This suggests that in this context the bottleneck for gene transfer should not be the release from the endocytotic vesicles, which is also in agreement with the results obtained by Kamata, et al., 1994 (see above).

The object was achieved according to the invention by a composition for the transfection of higher eukaryotic cells, the composition comprising a complex containing a nucleic acid to be expressed in the cell and, in a concentration which is suboptimal for the transfection, one or more cationic lipids and optionally helper lipid (e). The composition is characterized in that it contains one or more membrane-active acid peptides, the The ratio of the total number of positive to the total number of negative charges in the composition is approximately 0 to approximately 3.

Preferably, the ratio of positive to negative charges in the composition is from about 0 to about 2.

"Suboptimal concentration" is understood to mean the amount of cationic lipid at which the ratio of the positive charges of the cationic lipid to the negative charges of the nucleic acid is different from the ratio determined as optimal for the respective transfection, so that the ratio with cationic lipid, if appropriate with the addition of helper lipid, the transfection efficiency achieved is lower than at optimal ratios, "optimal" referring to the expression of the nucleic acid achieved as a result of the transfection (or in the case of using inhibiting RNA to the extent of the intended biological action) in the cell . The suboptimal concentration can be higher or lower than the optimal concentration.

The suboptimal concentration of cationic lipid, optionally in a mixture with helper lipid, preferably corresponds to a concentration at which the transfection efficiency is lower by a factor of at least about 2, preferably by a factor of about 5 to about 2,000, than at the optimal concentration.

The optimum concentration of cationic lipid for the respective transfection or the subobtimal concentration at which the transfection efficiency reaches at most half the value as at the optimal concentration depending on the cell type, * it can be determined in detail by titration, in that the kaionic lipid, which may be present in a mixture with helper lipid, is used in increasing (or also falling) concentrations; A reporter gene, for example a luciferase gene, is expediently used to determine the transfection efficiency.

Membrane-active peptides are defined by their ability to destabilize endosome membranes; they are also referred to as "endosomolytically active peptides", "endosome-breaking peptides" or as "fusogenic" peptides. These peptides have an amphipathic character and can form α-helices; due to their membrane-active properties, they are suitable for use in gene transfer methods in which the release of the genetic material transported into the cell from the endosomes is a limiting step, e.g. when importing nucleic acid into the cell via receptor-mediated endocytosis.

Suitable membrane-active peptides in the context of the present invention are peptides of natural origin or synthetic peptides, e.g. that in WO 93/07283, by Plank et al. 1994, or by Zauner et al. 1995, published peptides.

Whether peptides have suitable membrane-active properties and are therefore suitable for use in the context of the present invention can be determined with the aid of assays which simulate the process that takes place in the cell when endosomes are broken open. Suitable assays are the liposome and erythrocyte permeability assay, which have been described, for example, by Plank et al. , 1994. In a preferred embodiment, the composition contains a peptide called INF6 with the sequence GLF GAI AGFI ENGW EGMI DGWYG.

In a further preferred embodiment, the composition contains a peptide called INF10 with the sequence GLF ELA EGLA ELGW EGLA EGWYGC.

In a further preferred embodiment, the composition contains a peptide called INF5 with the sequence [GLF EAI EGFI ENGW EGnIDG] ₂ K.

In a further preferred embodiment, the composition contains a peptide of the name EGLA-I with the sequence GLFL GLA EGLA EGLA EGLA EGLA EGL EGLA GGSC.

In a further preferred embodiment, the composition contains a peptide called INFA with the sequence GLF EAI EAFI ENAW EAMI DAWYG.

Other suitable peptides are synthetic peptides of the designation INF8 with the sequence [GLF EAI EGFI ENGF EGMI DGGGJ2 K; the designation INF9 with the sequence GLF ELA EGLA ELGA EGLA EGWYGC; the name EGLA-II with the sequence WEA GLA EGLA EGLA EGLA EGLA EGL EGLA GGSC; the name EGLA-III with the sequence GLF EGA EGLA EGA EGLA EGLA EGWY GAC and the name EGLA-IV with the sequence GLF EGA EGLA EGW EGLA EGLA EGWY GAC.

Further synthetic membrane-active peptides suitable in the context of the present invention are those described by Plank et al., 1994, in particular peptides with the names INF4, INF4DI and INF7. In a further embodiment of the invention, the membrane-active peptide is modified with a lipid, for example with dipalmitoylphosphatidylethanolamyl (DPPE); modified and unmodified peptide may also be included in the composition. If a lipid-modified peptide is used, there is no need to add helper lipid.

In principle, the same lipids that are also used as helper lipids can be used as lipids for the modification of the membrane-active peptide; The choice of the lipid as well as the peptide is generally made for practical reasons, especially with regard to the coupling method, due to the presence of reactive groups. The components are coupled using methods known from the literature, e.g. as described by Martin et al. , 1989, or by Remy et al. , 1995.

The amount in which the optionally lipid-modified, membrane-active peptide is added to the transfection complex depends on the total positive charge of the lipid / DNA complex and on the total of the negative charges of the peptide and its molecular weight. The relative amount in relation to the cationic lipid (equivalents, given in mole peptide / mole cationic peptide) or the amount of absolute amount used is calculated in detail on the basis of these parameters. (In the case of INF6, for example, 2 charge equivalents of transfectam, corresponding to 6 nmoles, 5 μg INF6, corresponding to 2 nmoles, were used). With regard to a ratio of positive to negative charges of approx. 0 to approx. 3, preferably approx. 0 to approx. 2, the amount of peptide can initially be varied in preliminary tests, if necessary in comparison with other peptides, and in this way the optimally effective one Quantity can be determined. In principle, any mono- or polycationic lipid can be used in the context of the present invention, polycationic lipids being generally preferred. Numerous cationic lipids are known from the prior art which can be used as a constituent of the composition according to the invention. Examples of suitable cationic lipids are, for example, WO 95/02698, WO 91/16024, and the publications by Remy et al. , 1994; Solodin et al. , 1995; Feigner et al. , 1994; Ruysschaert et al., 1994; Weibel et al. , 1995; Le Bole'h et al. , 1995), to the disclosure of which reference is made.

Particularly preferred cationic lipids are lipopolyamines, e.g. those described in EP-AI 394 111, in particular DOGS (dioctadecylamidoglyclyspermin), which is available under the trade name "Transfectam".

As indicated above, the cationic lipid, optionally in a mixture with helper lipid, is contained in the transfection complexes in a suboptimal amount, ie the ratio between the positive charges of the lipid and the negative of the nucleic acid is greater or less than that used for the optimal gene transfer efficiency. taking into account the effect on transfection efficiency when defining helper lipid in the presence of helper lipid: for example, a helper lipid can improve the effect of cationic lipid at a concentration that would only have suboptimal efficiency without helper lipid, that the transfection achieved corresponds to that at an optimal lipid concentration; the The total concentration of the partners cationic lipid / helper lipid would be optimal in this case, despite the suboptimal concentration of cationic lipid alone.

"Helper lipids" are neutral lipids (natural or synthetic) that are physiologically or zwitterionic or free of charge, e.g. Cholesterol, dioleylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), phosphatidylglycerol, diacylglycerol, etc. Further examples of suitable helper lipids include described in WO 95/18863 and WO 95/02698, the disclosures of which are incorporated by reference.

According to the invention, the composition can contain one or more helper lipids.

Preferred helper lipids are the lipids DOPE, POPE, DOG (1,2-di-oleoyl-rac-glycerol), MOG (1-mono-oleoyl-rac ~ glycerol), EPC (egg phosphatidylcholine), EPE (egg phosphatidylethanolamine).

The helper lipids are preferably used in a concentration, based on the cationic lipid, of 0.1 to 10 equivalents (mol / mol).

The nucleic acids to be transported into the cell can be DNAs or RNAs, with no restrictions with regard to the nucleotide sequence. For gene therapy applications, DNA is primarily genes that are introduced into the cell, the expression of therapeutically effective gene products that are not or not sufficiently high in the cell be expressed to achieve. Inhibitory nucleic acids, for example antisense RNA molecules or ribozymes or the DNA molecules coding therefor, are also suitable for therapeutic purposes. Examples of nucleic acids which can be used in the context of the present invention are given, for example, in WO 93/07283 and in WO 95/18863.

In a further aspect, the invention relates to a method for transfecting higher eukaryotic cells, which is characterized in that the cells are brought into contact with the composition according to the invention.

The method can be used in in vitro, ex vivo or in vivo transfections, preferably for the transfection of mammalian cells. In vitro, the method is mainly used on cell cultures (adherent or in suspension). Applications ex vivo are gene therapy applications in which cells are removed from the organism to be treated and transfected ex corpore with a therapeutically active nucleic acid molecule, in order to subsequently be returned to the organism, where the gene product is expressed and has its therapeutic effect. An example of an ex vivo application is the production of tumor vaccines from autologous cells that are transfected with a cytokine gene.

For in vivo applications, the composition according to the invention is administered to the organism in the form of a pharmaceutical preparation which is also the subject of the present invention, preferably intravenously or, in the treatment of tumor diseases, intratumorally. For the pharmaceutical preparation, pharmaceutically acceptable additives and inert carriers, for example saline or phosphate-buffered saline or any carrier in which the compositions have suitable solubility properties can be added to the composition according to the invention. Regarding the formulation of pharmaceutical preparations, reference is made to Remington's Pharmaceutical Sciences, 1980.

The composition of the present invention can be modified to be used for applications with anionic molecules other than nucleic acid, e.g. to deliver anionic proteins to higher eukaryotic cells.

In the context of the present invention, it was surprisingly found that the transfection efficiency of the cationic lipid Transfectam (Behr, et al., 1989) when it was used with a small positive charge excess (1.5 and 2 charge equivalents), i.e. at suboptimal concentration, can be significantly enhanced by means of membrane-active acid peptides.

In the case of the peptide INF6 (2 μg / 1.5 charge equivalents transfectam), the amplification was up to 100,000 times, which corresponds to the level of gene expression achieved with optimal amounts of transfectam (high positive charge excess). Enhancement of gene expression with membrane-active peptides has been observed in several cell types. Among the membrane-active peptides tested, only the acidic ones gave good results in combination with Transfectam. This finding is in agreement with the hypothesis that the peptides for an improved release of the DNA from the endosomes in the same endocytotic vesicles must be localized like the lipid / DNA complexes, which is guaranteed when the peptides are ionically bound to the transfection particles. When using 2 charge equivalents of transfectam, the order of performance of the peptides was INF6>INF10> EGLA-I ^ INF5>INFA> Melittin. The results obtained for the peptides examined with Transfectam differ from the results obtained when the peptides were used in the transfection system based on the receptor-mediated endocytosis as a component of transfection complexes containing transferrin-polylysine: INF5 was the best peptide in this system and about 50 times more effective than INF6, while in combination with Transfectam it is 10 times less effective than INF6. The peptide INF5 has a membrane-breaking effect only at an acidic pH, while INF6 is membrane-active even at a neutral pH. In the lipid-free gene transfer system based on polylysine-conjugated cellular ligands, the activity of the peptide at neutral pH was accompanied by toxic side effects which were not observed when the peptide was used in the lipid system. The artificial peptide EGLA-I and INF10 were found to be more suitable as an addition to lipid / DNA complexes than complexes containing polylysine.

It was also found that the effect of the peptides tested on the transfection efficiency of cationic lipids at the sub-optimal charge ratio was of the same order of magnitude as the effect of helper lipids (Remy, et al., 1995; Zhou, et al., 1994; Feigner, et al ., 1994; Leventis, et al., 1990).

The transfection complexes containing cationic lipid and membrane-active peptide can be prepared by various methods. In the 30170 PCI7EP97 / 00649

14

Experiments of the present invention, the compositions were prepared by three different methods, the methods differing in the order of addition or combination of the complex components and in terms of the time of dilution. In the first variant, the DNA was added after the peptide was combined with transfectam; in the second variant, transfectam was first complexed with DNA and then the peptide was added. In the third variant, the peptide was added after dilution of the transfectam / DNA complex. It was examined whether the type of production of the transfection complexes has an influence on the extent of the increase in the transfection efficiency. It was shown that the production method affects the different peptides differently: For INF6, the three production methods proved to be equivalent. With INF5, the transfection complexes produced by the second and third methods gave better results, while with INF10 the first and third variants were superior to the second.

Regardless of the method of production, the combination of the mutant influenza peptides, which have an overall negative charge, with cationic lipids changes the state of charge of the transfection particles, with some of these peptide-containing complexes being close to electroneutrality because the mutant influenza apeptides have an overall negative charge, (see insert of Fig. 1).

In the experiments of the present invention, highly effective DNA complexes which contained the helper lipid DOPE at low charge (2 charge equivalents transfectam) were also included (Remy, et al., 1995) compared the membrane-active peptide-containing complexes of the present invention in terms of their transfection efficiency. After the enhancement by DOPE was attributed to its fusogenic activity (Allen, et al., 1990; Litzinger and Huang, 1992), the FACS data obtained in the context of the present invention (FIG. 3) indicate another important effect of the Helper lipids close: the cell association of the transfection complexes is much more pronounced if 1.5 equivalents of DOPE are added to 2 charge equivalents of Transfectam than if the lipospermin is used alone. It was shown that the membrane-active peptide did not increase the optimally effective complexes containing the helper lipid, whereas when using transfectam in excess (6 charge equivalents), i.e. when using a suboptimal amount, a significant increase in gene expression compared to that with optimal ones Amounts of transfectam alone were obtained.

The membrane-active peptide was also found to reduce the sensitivity of the transfection complexes to serum, which is important with regard to use in vivo.

Figure overview

Fig. 1: Transfection efficiency of Transfectam / DNA / INF6 complexes

Fig. 2: Effect of serum on transfection efficiency Fig. 3: A: flow cytometric analysis B: gene expression

Fig. 4: Effect of various membrane-active peptides on the transfection efficiency

5: Influence of the production method of membrane-active peptide-containing transfection complexes on the transfection efficiency

6: Efficiency of membrane-active peptide-containing complexes in the transfection of different cell lines

Fig. 7: Influence of bafilomycin AI on the transfection efficiency

Fig. 8: Influence of helper lipids on transfection efficiency

9: Influence of the combination of Transfectam,

Helper lipid and membrane-active peptide on transfection efficiency

10: Effect of a lipid-modified membrane-active peptide on the transfection efficiency

Unless otherwise stated, the following materials and methods were used in the following examples: a) Reporter gene plasmid pCMV-Luc

The construction of the plasmid pCMV-Luc, which carries the luciferase gene under the control of the CMV promoter / enhancer, was described under the name pCMVL in WO 93/07283.

b) Various reagents

The lipopolyamine DOGS (dioctadecylamidoglyclyspermin) with the trade name "Transfectam" was obtained from Promega, the helper lipids DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), MOG (1-mono-oleoyl-rac-glycerol), DOG (1.2 -di-0leoyl-rac-glycerol), EPE (egg phosphatidylethanolamine), EPC (egg phosphatidylcholine), as well as chloroquine, bafilomycin A ^ and melittin (from bee venom) were obtained from Sigma.

c) peptide synthesis

The name of the peptides, their origin and sequences are given in the table.

The INF5 and INF6 peptides were synthesized as described by Plank, et al. , 1994.

The INFA, INF10 and EGLA-I peptides were generated using the Fmoc strategy (N- (9-fluorenyl) methoxycarbonyl) with HBTU activation [0- (IH-benzotriazol-l-yl) -N, N, N ', N '- tetramethyluronium hexafluorophosphate], (Fastmoc ™ - 0.25 mmol scale) on an Applied Biosystems peptide synthesizer model 433 A (Foster City, California) with feedback monitoring. Three deprotection steps were carried out per cycle. If the feedback monitoring showed that the Fmoc deprotection was not quantitative, the next step was to double-couple the next amino acid and block the terminal NH2 groups (using acetic anhydride). The following amino acid protecting groups were used: (Trt) Asn, (Trt) Cys, (t-Bu) Cys, (t-Bu) Asp, (t-Bu) Glu, (Boc) Lys. A mixture of 70% NMP / 30% DMF was used as solvent.

For EGLA-I an HMP resin (Tentagel R PHB, 0.22 mmol / g, Rapp polymers) was used. The peptides were synthesized together until the rest of Gly-30, then half the amount of resin was used for the synthesis of EGLA-I.

The peptides INF10 and INFA were synthesized on a Cys (Trt) - preloaded aminomethylated polystyrene resin with a p-carboxytrityl chloride linker (0.52 mmol / g; PepChem, Tübingen, Germany) using DMF as solvent.

The INF7dimer peptide (a dimer of the INF7 peptide described by Plank et al., 1994) was synthesized using the Fmoc strategy on an Applied Biosystems peptide synthesizer model 431. A resin preloaded with cysteine was used (Tentagel S PHB-Cys). The medium-sized Lys DiFmoc was coupled as the first amino acid. Thereafter, double couplings were carried out up to Ile-18. Single couplings were performed from Met-17 to Ile-10; with Phe-9 a single coupling, then additional coupling with 1% Triton in the coupling approach. From Gly-8, simple pairings were carried out.

Deprotection of the peptides and cleavage from the resin were carried out using a mixture of phenol, ethanedithiol, thioanisole, water and trifluoroacetic acid (0.75: 0.25: 0.5: 0.5: 10). The crude peptides were precipitated by dropwise addition to ether and centrifuged. The peptides thus obtained were washed three times with ether and then dried under a stream of argon, followed by high vacuum. The crude peptides were dissolved in 1 M TEAB, pH 9 and 1% β-mercaptoethanol.

The purity of the peptides was determined by means of analytical reverse phase HPLC using a C-18 column (Vydac 218ATP54, 2.1 mm x 25 cm, 5 μm). A binary solvent system (solvent A: aqueous 0.1% trifluoroacetic acid; solvent B: acetonitrile containing 0.1% trifluoroacetic acid) was used at a gradient of 0-100% in 45 minutes at a flow rate of 1 ml / min. Analytical ion exchange chromatography (SuperQ-Toyopearl 650, TosoHaas, 5 mm x 50 mm column) was carried out using a salt gradient (20 mM HEPES, pH 7.3, 0-1.5 M NaCl in 60 min, flow rate 0.5 ml / min).

The EGLA-I peptide was subjected to gel filtration (Sephadex G10, 20 mM TEAA, pH 7.3). The purified peptide fraction was freeze-dried in a Speedvac (Savant). The analytical reverse phase chromatography showed a purity of approx. 95%. The solutions of the raw peptides INF10 and INFA were fractionated by gel filtration on Sephadex G10 (10 mm x 300 mm column) in 20 mM TEAA, pH 7.3. The peptides were lyophilized; analytical reverse phase chromatography showed a purity of approx. 98 and 95%, respectively.

The peptide INF7dimer was purified over Sephadex G 10 (buffer HBS; column 10mm x 300mm). Ion exchange chromatography was then carried out (column: (10 mm × 100 mm); material: Toso Haas Super Q 650 S; flow rate: 0.5 ml / min; eluent: A: 20 mM Hepes 7.3, B: 3 M NaCl / 20 mM Hepes 7.3; Gradient: 0 - 40 min 0% B, 40 - 140 min 100% B, ie 1% / min). The peptide eluted at 70-80 min. The peptide purified via ion exchanger was again cleaned on a Sephadex G 10 column (10mm x 300mm) (buffer HBS / 50% glycerin).

The identity of the peptides was determined as described by Plank, et al. , 1994. The peptides were frozen in liquid nitrogen.

The liposome leakage assay was performed as described by Plank, et al. , 1994.

d) cell culture and media

The media, fetal calf serum (FCS) and horse serum were purchased from Gibco-BRL. The culture media were supplemented with 2 mM L-glutamine and antibiotics. Primary human melanoma cells of the designation H225, mouse embryo liver cells of the cell line TIB-73 (ATCC BNL CL.2), human lung carcinoma cells of the cell line A549 (ATCC CCL 185) and cells of the mouse melanoma cell line Cloudman S91, clone were used as cells M3 (ATCC No. CCL 53.1) used. The H225 cells were in RPMI 1640/10% FCS / 1 mM sodium pyruvate, the A549 cells in DMEM / 10% FCS, the BNL CL.2 cells in high-glucose DMEM / 10% FCS and the M3 cells in Ham's F10 medium / 15% horse serum / 5% FCS grown.

e) transfection of the cells

i) Preparation of cationic lipid / DNA complexes

The complexes were prepared as described by Barthel, et al. , 1993, where 3 μg of plasmid DNA and the amount of transfectam used in each case were dissolved in 75 μl of 150 mM NaCl. After 10 to 20 minutes, the two solutions were combined. After a further 10 min the mixture was diluted to a total volume of 2 ml with serum-free medium.

The term "charge equivalent" indicates the amount of cationic lipid used for transfection; 1 charge equivalent corresponds to the amount required to neutralize all negative charges of the phosphate groups of the plasmid. 3 μg of DNA correspond to 9 nmoles of negative charges; when calculating the charge ratio, it was taken into account that 1 mole of transfectam has 3 moles of positive charges which originate from the three ammonium groups protonated at physiological pH. Starting from the molecular weight of approx. 1250, this means that when using 3 μg DNA for a double positive charge excess (2 charge equivalents

Transfectam / 3 μg DNA) 3 μl of a 2mM transfectam solution containing 6nmol (= 7.58 μg) transfectam required.

The peptides were contained in an amount of 0.5 or 1 mg / ml solution in HBS (HEPES-buffered saline) 150 mM NaCl, 20 mM HEPES pH 7.3) to the

Given transfectam / DNA complexes. After a 10 to

That was 20 minutes of ripening

Transfection volume brought to 2 ml with culture medium, 1 ml of the transfection mixture obtained was pipetted onto the cells.

The helper lipids DOPE, DOG, EPC, EPE or MOG or cholesterol were diluted in ethanol containing a trace of dichloromethane. The

Transfectam / helper lipid / DNA complexes were formed by mixing the respective amounts of transfectam / helper lipid (the amount of helper lipid used, based on transfectam, is given as a molar ratio) before dilution with the DNA solution.

ii) transfection and luciferase determination

50,000 to 75,000 cells per well of a 24-well plate were plated one day before the transfection, the transfection volume being 1 ml in all experiments. The transfection was carried out with 1 ml of the complex prepared in i). After 3 to 4 hours the transfection medium was replaced by fresh medium containing 10% FCS. Unless otherwise stated, each experiment was carried out at least twice, the amounts of peptides shown in the figures in these cases relating to the total amount used for the double determination.

24 hours after the transfection, the cells were harvested and taken up in 150-200 μl 250 mM Tris pH 7.3, 0.5% Triton X-100. The cellulose was then transferred to 1.5 ml Eppendorf tubes and centrifuged at 14,000 g for 5 min in order to close the cell debris pelletize. The determination of the luciferase activity was carried out as described in WO 93/7283, the luciferase light units being determined from an aliquot of the supernatant (20 μl) with 10 seconds integration after injection of freshly prepared luciferin solution. The luciferase background (150-250 light units) was subtracted from each value; the transfection efficiency was expressed as total light units, which represent the mean of duplicate determinations. The Bradford assay (Bio-Rad) was used for quantitative determination of the protein content.

f) flow cytometry

For flow cytometry, the plasmid DNA was incubated with the intercalating fluorescent dye YOYO-1 (described by Rye, et al., 1992; Hirons, et al., 1994, available from Molecular Probes; approx. 1 dye molecule per 300 bp). the complexes were then prepared as described above. The complexes were placed on 300,000 H225 cells per well of a 6 well plate, then incubated for 4 hours either at 4 ° C (cell surface association) or at 37 ° C (cell surface association and uptake into the cells). The cells were then washed twice with cold PBS and harvested with 1 mM EDTA in PBS and analyzed on a FACScan device (Becton Dickinson).

example 1

Transfection efficiency of Transf ec camo / DNA / INF6 complexes Complexes of 1, 1.5, 2 or 4 charge equivalents of transfectam per 3 μg pCMV-Luc in combination with increasing amounts of the membrane-active, acidic peptide INF6 derived from the influenza virus were prepared and mixed with RPMI 1640 culture medium. The complexes obtained were applied to H225 cells (75,000 cells per well of a 24-well plate). After 4 h, the transfection medium was replaced by fresh RPMI medium containing 10% FCS. The result of the experiments is shown in FIG. 1 (dotted line with rhombuses: 1 equivalent (eq.) Transfectam / x μg INF6; full line with circular. Transfectam 1.5 eq./x μg INF6; dashed line with squares: Transfectam 2 eq./x μg INF6; full line with triangles: Transfectam 4 eq./x μg INF6). It was found that when 2 or 1.5 charge equivalents of lipid were used, a 100 to 1,000 fold increase in transfection efficiency, measured as luciferase activity, was achieved. When using optimal conditions (4 charge equivalents of cationic lipid), only a slight increase (approx. 1.5 to 5-fold) was obtained. Since the INF6 peptide has four negative charges at pH 7, it can associate with the cationic lipid / DNA particles via electrostatic interactions. No enhancement was observed when using neutral particles (1 charge equivalent), presumably because only a few peptides can bind to these complexes. Interactions with the cell membrane could even be reduced because positive residual charges are masked by the association with the peptide. The insert of FIG. 1 shows the transfection efficiency of the transfectam / DNA / INF6 complexes, plotted against the theoretical charge ratio of the complexes. Assuming that almost all of the peptide binds to the transfecting particles, a high Luciferase expression achieved with electroneutral (or almost neutral) lipid vectors.

Example 2

Effect of serum on transfection efficiency

Transfections of H225 cells were carried out using the amounts of transfectam (in charge equivalents) shown in FIG. 2 and the peptide INF6. The transfections were carried out on the one hand without serum (empty bars), on the other hand in the presence of 10% (dotted bars). 20% (gray bars) FCS not heat-inactivated (Fig. 2). The peptide-containing complexes were found to be less sensitive to serum.

Example 3

Association of transfectam / DNA complexes and expression rate

a) Flow cytometry analysis of transfection complexes

The analysis was carried out with H225 cells as described in the method section. As shown in FIG. 3A, the association of the transfectam / DNA complexes with the cell surface changes decisively with the charge ratio: a heterogeneous cell population was found with 2 charge equivalents, while a classic Gaussian curve was found with 4 charge equivalents. The addition of membrane-active peptides to 2 (or 4) charge equivalents resulted in transfectam no significant change in the curve profile at both 4 ° C and 37 ° C.

b) gene expression

In parallel to the tests carried out in a), the gene expression of 300,000 cells after incubation at 4 ° C. and 37 ° C. with different

Transfection complexes were measured (the cells were incubated at 4 ° C. or at 37 ° C., washed twice with PBS after 4 h, mixed with fresh medium containing 10% FCS and incubated at 37 ° C. and 5% CO 2 for 20 h). The presence of peptide (1.5 μg / well; in this experiment a single determination was carried out) caused a 15-fold increase in expression when the mixture was prepared at 4 ° C. and a 100-fold increase in the mixture at 37 ° C. (Fig. 3B. Empty bars: gene expression after incubation at 4 ° C; filled bars: gene expression after incubation at 37 ° C).

Example 4

Effect of different peptides

As described in the method section, transfeetarn / DNA complexes were prepared from 2 charge equivalents of transfectam per 3 μg DNA. For some experiments, the complexes contained the peptides indicated in FIG. 4 in the amounts indicated in each case. The peptides INF6, INFA and melittin, which have good hemolytic activity without a pronounced specificity for low pH values, showed different behavior: INFA and INF6 increased the transfection efficiency of 2 charge equivalents of transfectam by 10 and 200 times, respectively Melittin only slightly increased luciferase expression. In addition, melittin was highly toxic at 5 μg / double determination. The influenza apeptide mutants INF5 and INF10, which had been shown in the liposome permeability test to release calcein efficiently at pH 5.0 (Plank, et al., 1994) gave better results than INFA, but were less effective than INF6. In addition, a peptide called EGLA-I, which was not derived from the HA2 sequence of the influenza virus, was tested, in which one of the alanine residues in the GALA repeat (Parente, et al., 1988) was replaced by a glycine residue. This peptide gave results as good as INF5.

Example 5

Influence of the manufacturing method of

Transfectam / DNA / peptide complexes on the efficiency of transfection of H225 cells

Complexes were produced in three different ways (FIG. 5): a) Combination of peptide and lipid before complexation with DNA (dotted bars) b) Addition of the peptide after formation of the transfectam / DNA complex before dilution with medium (150 μl Volume; gray bars) c) adding the peptide after dilution of the complexes with the culture medium (2 ml volume; black bars)

As the results shown in FIG. 5 show, the influence of the production method on the gene expression rate depends on the peptide sequence. Combining the peptide with Transfectam before complexing with the plasmid gave a contrary result 30170 PCΪ7EP97 / 00649

28

Behavior of the peptides: while the gene expression rate remained unchanged at INF6, the performance of the complexes produced in this way was five times better than that obtained with the standard method (see method part). In contrast, this method of complex formation in INF5 caused a sharp reduction in gene expression.

Example 6

Effect of the peptides on the transfection efficiency of different cell lines

The fact that the effect of the peptides on gene transfer by means of lipids is not restricted to certain cell types, but rather a general phenomenon, was shown in experiments with cells of different cell lines, which are shown in FIG. 6 (H225 cells: empty bars; BNL CL.2 Cells: dotted bars; A549 cells: light gray bars; M3 cells: dark gray bars). For this purpose, complexes were made from Transfectam, pCMV-Luc 5 μg INF6 and the efficiency of the complexes was tested on the cells. (As a comparison value, transfectam was used with a charge excess of 4 charge equivalents, although there are not the highest values on all cells tested.) Increase rates of the transfection efficiency were found for all cell lines if the transfection complexes contained a membrane-active peptic.

Example 7

Investigation of the influence of bafilomycin A on the transfection efficiency of transfectam / DNA complexes The presence of the specific inhibitor of the vacuole proton pump bafilomycin A ^ (Bowman, et al., 1988; Yoshimori, et al., 1991) at a concentration of 200 nM in the transfection of H225 cells (2 charge equivalents of transfectam or

2 charge equivalents of Transfectam plus INF10 or INF6 used; after 4 h of incubation, the medium was replaced by fresh one with a content of 10% FCS), the gene expression decreased by a factor of 7, 5 or 1.5 (FIG. 7). Empty bars: without addition of bafilomycin AI. Dotted bars: in the presence of 200 nM bafilomycin AI). When using 4 charge equivalents of transfectam, bafilomycin was unable to reduce the gene transfer.

Example 8

Investigation of the influence of helper lipids on the efficiency of transfectam / DNA complexes

a) Enhancing the effect of transfectam with helper lipids

The helper lipids shown in FIG. 8 were used in a molar ratio of 2 charge equivalents of transfectam to 1 to 3 equivalents of helper lipid. For DOPE, EPE or DOG, this resulted in a gene expression that was 10 to 20 times higher (see also FIG. 3B). MOG increased expression approximately 4-fold, while EPC and cholesterol were unable to increase transfection efficiency on H225 cells. A further, up to 7-fold increase could be achieved if 1 mol /% DOG was added to the transfectam / DOPE formulation; this is presumably due to the ability of DOG to induce fusion, among other things (Siegel, et al., 1989).

A flow cytometry was carried out as described in Example 3a) with transfection complexes containing the helper lipid DOPE (1.5 mol per 2 mol transfectam; that is 9 nmol DOPE, corresponding to 6.7 μg). There was a significant change in the FACScan profile (FIG. 3A): the cell population is more homogeneous and the cell association (and also the uptake into the cells at 37 ° C., not shown in the figure) is stronger than without the helper lipid. When the luciferase activity from an experiment carried out at 4 ° C, in which 2 charge equivalents of transfectam and 1.5 equivalents of DOPE were measured 24 hours later, it was found that the complexes containing DOPE were 8 and 280 times better than 4 and 2 charge equivalents, respectively of the cationic lipid alone (Fig. 3B). When the experiment was carried out at 37 ° C, the differences in luciferase expression were 2 and 640 times, respectively.

b) enhancement of the effect of transfectam by helper lipids and membrane-active peptides

After the production of Transfectam / DNA / DOPE complexes, increasing amounts of INF10 were added. After a maturation time of 10-20 min, serum-free culture medium was added to a total volume of 2 ml; 1 ml of the transfection mixture was applied to each well of the duplicate assay. The result of these tests is shown in FIG. 9 (empty bars: transfectam, dotted bar: Transfectam 6 eq.Transfectam / DOPE 0.36 eq. , filled

Bars: Transfectam 6 eq.Transfectam / DOPE

0.36 eq./x μg INF10). The presence of membrane active

Peptide resulted in complexes with high efficiency

(Transfectam 2 eq./INFlO 3 μg, 5 μg or

7.5 μg / 1.5 eq. DOPE) no further increase (not in

Fig. 9 shown). However, when the peptide was used together with an excess of transfectam (6 charge equivalents), it could be compared to that with optimal

Amounts of Transfectam / DOPE values obtained will give a significant increase in gene expression; the values with

6 eq.Transfectam / 0.36 eq. DOPE / 10 μg INF10 were 6x better than those with

2 eq.Transfectam / 1.5 eq. DOPE / 7.5 μg INF10.

Example 9

Effect of a lipid-modified membrane-active peptide on transfection efficiency

a) Production of modified peptide (DPPE-INF7dimer)

The peptide with the designation INF7dimer (see table), a dimer of that described by Plank, et al. , 1994 described peptide INF7, was attached to the lipid derivative DPPE-

(Dipalmitoylphosphatidylethanolamyl) bromoacetamide

(Hexadecanoic acid 3- ((2- (2- (2- (2- (2- (2-bromoacethylamino) ethoxy) ethoxy) - acetylamino) ethoxy) hydroxyphosphoryloxy) -2-hexadecanoyloxypropyl ester) coupled by 0.95 equivalents of peptide (80 nmol; approx. 450 μg) were added to the lipid diluted in 1.3 ml diethanolamine buffer (pH 9.5 / ethanol; 9/1; v / v). After 3 h at room temperature an Ellman test became quantitative Determination of the thiol residues carried out. Then β-mercaptoethanol was added to destroy the remaining bromoacetamide group. The lipid-modified peptide obtained was used without further purification.

b) Transfection of BNL CL.2 cells

1.5 eq. Transfectam were diluted in 75 μl 0.15 M NaCl, mixed intensively, whereupon the amounts of DPPE-INF7dimer indicated in FIG. 10 (the values given in the figure are mol%, based on the total amount of transfectam) were added. The mixture was then mixed well, after 10 min 3 μg DNA in 75 μl NaCl were added, mixed again and after a further 10 min filled with medium to a total transfection volume of 2 ml. 1 ml per well of the transfection composition obtained was applied to the cells. The results shown in Fig. 10 show that the highest increase was obtained at a content of 7.5 mol% DPPE-INF7dimer.

table

Sequences and origin of membrane-active peptides

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Claims

claims

1. Composition for the transfection of higher eukaryotic cells, the composition containing a complex containing a nucleic acid to be expressed in the cell and, in a suboptimal concentration for the transfection, one or more cationic lipids and optionally helper lipid (s) characterized in that the composition contains one or more membrane-active acid peptides, the ratio of the total number of positive to the total number of negative charges in the composition being about 0 to about 3.

2. Composition according to claim 1, characterized in that the ratio of the positive to the negative charges of the composition is about 0 to about 2.

3. Composition according to claim 1, characterized in that the suboptimal concentration of cationic lipid, optionally in a mixture with helper lipid, corresponds to a concentration at which the transfection efficiency is a factor of at least about 2 less than at an optimal concentration.

4. Composition according to one of claims 1 to 3, characterized in that it contains a peptide called INF6 with the sequence GLF GAI AGFI ENGW EGMI DGWYG.

5. Composition according to one of claims 1 to 3, characterized in that it contains a peptide of the designation INF10 with the sequence GLF ELA EGLA ELGW EGLA EGWYGC.

6. A composition according to any one of claims 1 to 3, characterized in that it comprises a peptide labeled with the sequence INF5 [GLF EAI EGFI ENGW EGnIDG] ₂ K.

7. Composition according to one of claims 1 to 3, characterized in that it contains peptide of the name EGLA-I with the sequence GLFL GLA EGLA EGLA EGLA EGLA EGL EGLA GGSC.

8. Composition according to one of claims 1 to 3, characterized in that it contains a peptide of the name INFA with the sequence GLF EAI EAFI ENAW EAMI DAWYG.

9. A composition according to any one of claims 1 to 3, characterized in that it comprises a peptide labeled with the sequence INF8 [GLF EAI EGFI ENGF EGMI DGGG] ₂ K.

10. Composition according to one of claims 1 to 3, characterized in that it contains a peptide with the reference INF9 with the sequence GLF ELA EGLA ELGA EGLA JWYGC.

11. The composition according to any one of claims 1 to 3, characterized in that it contains a peptide of the name EGLA-II with the sequence WEA GLA EGLA EGLA EGLA EGLA EGL EGLA GGSC.

12. Composition according to one of claims 1 to 3, characterized in that it contains a peptide of the name EGLA-III with the sequence GLF EGA EGLA EGA EGLA EGLA EGWY GAC.

13. Composition according to one of claims 1 to 3, characterized in that it contains a peptide of the name EGLA-IV with the sequence GLF EGA EGLA EGW EGLA EGLA EGWY GAC.

14. Composition according to one of claims 1 to 3, characterized in that it contains a peptide called INF7dimer with the sequence [GLF EAI EGFI ENGW EGMI DGWYG] ₂ KC.

15. Composition according to one of the preceding claims, characterized in that the membrane-active peptide is modified with a lipid.

16. The composition according to claim 15, characterized in that the peptide is modified with dipalmitoylphosphatidylethanolamyl (DPPE).

17. Composition according to one of the preceding claims, characterized in that the cationic lipid is a lipopolyamine.

18. The composition according to claim 17, characterized in that the lipopolyamine is dioctadecylamidoglyclyspermin (DOGS).

19. Composition according to one of the preceding claims, characterized in that it contains one or more helper lipids selected from the group Group phosphatidylethanolamines,

Contains phosphatidylglycerols and diacylglycerols.

20. The composition according to claim 19, characterized in that it contains dioleylphosphatidylethanolamine (DOPE) as helper lipid.

21. The composition according to claim 19, characterized in that it contains oleoyl-palmitoylphosphatidylethanolamine (POPE) as helper lipid

22. The composition according to claim 19, characterized in that it contains 1-mono-oleoyl-rac-glycerol (MOG) as helper lipid

23. The composition according to claim 19, characterized in that it contains 1,2-di-oleoyl-rac-glycerol (DOG) as helper lipid.

24. The composition according to claim 19, characterized in that it contains egg phosphatidylcholine (EPC) as helper lipid.

25. The composition according to claim 19, characterized in that it contains egg phosphatidylethanolamine (EPE) as helper lipid.

26. The composition according to claim 19, characterized in that it contains cholesterol as helper lipid.

27. A pharmaceutical preparation containing as active component a composition according to any one of claims 1 to 25.

28. A method for transfecting higher eukaryotic cells, characterized in that the cells are brought into contact with a composition according to one of claims 1 to 26.

29. Application of the method according to claim 28 to mammalian cells in vitro or ex vivo.