DE19605548A1 - Composition for transfection of higher eukaryotic cells - Google Patents

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

The invention relates to the transfection of higher eukaryotic cells, especially of Mammalian cells.

Need for an efficient system for introduction of nucleic acid in living cells consists mainly in Framework of gene therapy. This involves genes in cells introduced to therapeutically synthesize in vivo to achieve more effective gene products.

The most advanced and so far common technologies for the application of Use nucleic acids in the context of gene therapy Systems for the transfer of genes into the cell work on the basis of viruses, in particular Retroviruses and adenoviruses (Miller, 1992; Mulligan, 1993; Berkner; 1988), and cationic lipids (Behr, 1994). Alternative strategies for gene transfer are based on mechanisms, the cell for which Served transport of macromolecules. An example for 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-A1 0 388 758).

Of the synthetic ones developed in the last decade Gene transfer vehicles have become mono- and polycationic amphipathic lipids, the DNA can complex and condense as special promising (Behr, et al., 1989; Felgner, et al., 1987 and 1994; Leventis, et al., 1990; Gao, et al., 1991; Rose, et al., 1991; Mawley-Nelson, et al., 1993; Weibel, et al., 1995; Solodin, et al., 1995).  

Efforts to improve the performance of Gene transfer methods based on cationic So far, lipids have focused on the direct Modification of the cationic lipid, e.g. B. by Acyl groups, spacer arms or hydrophilic sections (Remy, et al., 1994; Felgner, et al., 1994). Despite improved results due to such measures still a lot about the entry mechanism of Lipid / DNA particles in the cell unclear. Because of recent publications (Zabner, et al., 1995) the main transport route for these particles is likely to be via the Endocytosis.

Cationic lipids show the best in vitro Transfection results with a clearly positive Excess charge. So z. B. for lipospermin DOGS (Dioctadecylamidoglyclyspermin, commercial available under the trade name "Transfectam") three to six times the excess of positive charges in relation to DNA than for that Transfection efficiency optimally proven (Barthel, et al., 1993). The positive charges promote binding of the complex and its uptake into the cells. In addition, Transfectam has a buffering effect on the Endosomes (the pKa of the least basic secondary amine of lipospermin that for the Buffer effect is decisive is about 5.4; Behr, 1994) and therefore protects DNA on the one hand enzymatic degradation, on the other hand, it causes a osmotic swelling and a subsequent one Destabilization of the buffered endosomes. A strong one thus positive charge of the lipid / DNA complexes the application in vivo u. a. the disadvantage that the Lipid / DNA particles only have a very short half-life to have.  

To the efficiency of Lipopolyamine DOGS and DPPES (Palmitoylphosphatidylethanolamine) at low positive excess charge (ratio positive: negative charges 2, preferably 0.5 to 1.5) to improve, was the addition of adjuvants proposed that with the Lipopolyamin / nucleic acid Kom can associate plex, e.g. B. the addition of so-called "Helper Lipids".

Kamata, et al., 1994, reviewed the efficiency of the Gene transfers made with Lipofectin, a 1: 1 mixture the monocationic lipid DOTMA (N- [1- (2,3-dioleyl oxy) propyl] -N, N, N-trimethylammonium chloride) and the Helper lipid DOPE (dioleylphosphatidylethanolamine), at a positive charge surplus of 1.25 was increased by 3 to 5 times using peptides.

Similarly, it was proposed in WO 95/02698 A1 for which Transfection of cells in cationic lipids Combination with membrane-active peptides from viruses with Shell, u. a. of influenza viruses, where one of the Kamata et. al., 1994 described peptides or one of the glycoprotein of vesicular stomatitis Virus derived peptide in combination with Lipofectamine, a 3: 1 mixture of the polycationic Lipids 2,3-dioleyloxy-N- [2 (spermincarboxamido) ethyl] - N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and DOPE used and with one determined as optimal positive excess charge of approx. 20 a Improve transfection efficiency by up to Was achieved 12 times.

On the other hand, brought cationic lipids with viral peptides were conjugated to give them the fusogenic or karyophilic properties of viruses  bestow disappointing results at optimal Lipid concentration (Remy et al., 1995).

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

When solving the task posed first asked the question if it was the endocytosis subsequent steps are how to transport DNA from the endosome into the cytoplasm and further into the nucleus, which the bottleneck for a successful gene transfer with represent cationic lipids.

It was found that the addition of membrane-active influenza apeptides only a minor one Enhancement of gene expression (1.5 to 5 fold) with Transfectam, if this works at optimal Concentrations, so with a high positive Excess charge, is used. This suggests conclude that the bottleneck for the gene transfer does not release from the endocytotic vesicles, which should also be in the Consistent with those obtained from Kamata, et al., 1994 Results is available (see above).

The object was achieved by a Composition for the transfection of higher eukaryotic cells dissolved, the composition a complex containing one in the cell too expressing nucleic acid as well, in one for the Transfection suboptimal concentration, one or several cationic lipids, as well as optionally Helper lipid (s). The composition is characterized in that they have one or more contains membrane-active acid peptides, the  Ratio of the total number of positive to the total number of negative charges in the composition is about 0 to about 3.

The ratio of the positive to is preferably the negative charges in the composition about 0 to about 2.

Under "suboptimal concentration" the amount of cationic lipid understood at which the ratio the positive charges of the cationic lipid to the negative charges of the nucleic acid is different from that is optimal for the respective transfection determined ratio, so that with cationic Lipid, optionally with the addition of helper lipid, transfection efficiency achieved is lower than in optimal conditions, with "optimal" on the expression achieved as a result of the transfection Nucleic acid (or in the case of using inhibitory RNA to the extent of the intended biological effect) 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 a concentration at which the Transfection efficiency by a factor of at least approx. 2, preferably by a factor of approx. 5 to approx. 2000, is less than with optimal concentration.

The optimal one for each transfection Concentration of cationic lipid or the subobtimal concentration at which Transfection efficiency at most half the value achieved as with optimal concentration  depending on cell type; it can be done in detail by titration can be determined by the cationic lipid, the optionally in a mixture with helper lipid, in increasing (or falling) concentrations is used; is useful for determining the Transfection efficiency a reporter gene, e.g. B. a Luciferase gene used.

Membrane active peptides are by their ability defined to destabilize endosome membranes; she are also called "endosomolytically active peptides", "Endosome-breaking peptides" or as "fusogenic" Designated peptides. These peptides have amphipathic Character and can form α-helices; they are due to their membrane-active properties for the Suitable for use in gene transfer methods in which the Release of the transported into the cell genetic material from the endosomes represents limiting step, e.g. B. when importing Nucleic acid in the cell via receptor-mediated Endocytosis.

As membrane-active peptides in the context of the present Peptides of natural origin are suitable according to the invention or synthetic peptides, e.g. B. those in WO 93/07283 A1, by Plank et al. 1994, or by Zauner et al. 1995, published peptides.

Whether peptides have suitable membrane-active properties have and thus for use in the context of present invention can be considered with Assays are used to determine the process simulate the breakup in the cell Endosomes expires. Suitable assays are liposome and the erythrocyte permeability assay, e.g. B. by Plank et al., 1994.  

In a preferred embodiment, the Composition of a peptide called INF6 with the Sequence GLF GAI AGFI ENGW EGMI DGWYG.

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

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

In a further preferred embodiment contains the composition is a peptide called EGLA-I with the sequence GLFL GLA EGLA EGLA EGLA EGLA EGLEGLA GGSC.

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

Other suitable peptides are synthetic peptides Designation INF8 with the sequence [GLF EAI EGFI ENGF EGMI DGGG] ₂ K; called 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; of the Name EGLA-III with the sequence GLF EGA EGLA EGA EGLA EGLA EGWY GAC and the designation EGLA-IV with the Sequence GLF EGA EGLA EGW EGLA EGLA EGWY GAC.

Other suitable within the scope of the present invention synthetic membrane-active peptides are those from Plank et al., 1994, described, in particular peptides of Designation INF4, INF4DI and INF7.  

In a further embodiment of the invention that is membrane active peptide modified with a lipid, e.g. B. with dipalmitoylphosphatidylethanolamyl (DPPE); it can also be modified and unmodified Peptide can be included in the composition. At Use of a lipid modified peptide can be based on the addition of helper lipid can be dispensed with.

As a lipid for the modification of the membrane-active Peptides can basically use the same lipids that are also used as helper lipids; the lipid as well as the peptide are selected in general, for practical reasons, with regard to the coupling method, due to the presence of reactive groups. The coupling of the components takes place according to methods known from the literature, e.g. B. as from Martin et al., 1989, or from Remy et al., 1995, described.

The amount in which the, optionally lipid-modified, Membrane-active peptide added to the transfection complex is dependent on the positive total charge of the Lipid / DNA complex as well as the sum of the negative Charges of the peptide and its molecular weight. The relative amount in relation to the cationic lipid (Equivalents given in moles of peptide / moles of cationic Peptide) or the amount of absolute amount used, is calculated in detail based on these parameters. (In the case of INF6, for example, for 2 charge equivalents Transfectam, corresponding to 6 nmoles, 5 µg INF6, corresponding to 2 nmoles, used). With regard to a Ratio of positive to negative charges of approx. 0 to about 3, preferably about 0 to about 2, can Peptide amount first in preliminary tests, if necessary in Comparison with other peptides, varies and on this Way the optimally effective amount can be determined.  

In principle, within the scope of the present invention any mono- or polycationic lipid is used be polycationic lipids in general to be favoured. Are from the prior art numerous cationic lipids known as Part of the composition according to the invention can be used. Examples of suitable ones cationic lipids are e.g. B. WO 95/02698 A1, the WO 91/16024 A1, as well as the publications by Remy et al., 1994; Solodin et al., 1995; Felgner et al., 1994; Ruysschaert et al., 1994; Weibel et al., 1995; Le Bole′h et al., 1995), to the disclosure of which reference is taken.

Cationic lipids are particularly preferred Lipopolyamines, e.g. B. in EP 394 111 A1 described, especially DOGS (Dioctadecylamidoglyclyspermin), which under the Tradename "Transfectam" is available.

The cationic lipid, optionally in a mixture with Helper lipid, as indicated above, in the Contain transfection complexes in suboptimal quantities, d. H. the ratio between the positive charges of the Lipids and the negative of the nucleic acid is larger or less than that for the optimal Gene transfer efficiency used, whereby for the Definition of the suboptimal amount of cationic lipid in the presence of helper lipid, its effect the transfection efficiency is also taken into account: It can e.g. B. a helper lipid the effect of cationic lipid at a concentration without Helper lipid would only have suboptimal efficiency, so improve the transfection achieved by those at optimal lipid concentration; the  Total concentration of the partners cationic Lipid / helper lipid would be in this case, despite suboptimal concentration of cationic lipid alone, optimal overall.

"Helper lipids" are neutral lipids (of natural origin or synthetic) which under physiological conditions zwitterionic or free of charges, e.g. B. Dioleylphosphatidylethanolamine (DOPE), oleoyl-palmitoylphosphatidylethanolamine (POPE), Phosphatidylglycerols, diacylglycerols, etc. Others Examples of suitable helper lipids include a. in the WO 95/18863 A1 and described in WO 95/02698 A1 the disclosures of which are referred to.

According to the invention, the composition can be a or contain several helper lipids.

Preferred as 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 in one Concentration, based on the cationic lipid, of 0.1 to 10 equivalents (mol / mol) are used.

The nucleic acids to be transported into the cell can be DNAs or RNAs, where no restrictions on the nucleotide sequence consist. The DNA is for gene therapy applications especially around therapeutically effective genes that enter the cell be introduced to therapeutic expression more effective gene products that are not in the cell or not expressed in a sufficiently high concentration  are going to achieve. Come for therapeutic purposes also inhibitory nucleic acids, e.g. B. Antisense RNA molecules or ribozymes or the ones therefor coding DNA molecules. examples for Nucleic acids within the scope of the present invention can be used, for. B. in the WO 93/07283 A1 and WO 95/18863 A1.

In a further aspect, the invention relates to a Process for transfection of higher eukaryotic Cells, which is characterized in that the Cells with the composition according to the invention in Touches.

The method can be used in vitro, ex vivo or in vivo Transfections are used, preferably for the Transfection of mammalian cells. In vitro it will Process mainly on cell cultures (adherent or in Suspension) applied. Applications are ex vivo gene therapy applications in which the Treating organism cells taken and ex corpore with a therapeutically effective nucleic acid molecule, be transfected and then returned to the Organism to be returned where the gene product is expressed and its therapeutic effect unfolded. An example of an ex vivo application is the production of tumor vaccines from autologous cells, that are transfected with a cytokine gene.

For applications in vivo, the organism is composition according to the invention in the form of a pharmaceutical preparation, also subject of the present invention is administered preferably intravenously or, in the treatment of Tumor diseases, intratumoral.  

For pharmaceutical preparation, the pharmaceutical composition according to the invention acceptable additives and inert carriers, e.g. B. Saline or phosphate buffered saline or any carrier in which the compositions have suitable solubility properties added will. Regarding the formulation of pharmaceutical Preparations are made at Remington’s Pharmaceutical Sciences, 1980.

The composition of the present invention can modified to be used for applications on others anionic molecules used as nucleic acid be, e.g. B. anionic proteins, in higher to promote eukaryotic cells.

In the context of the present invention was surprising found that the transfection efficiency of the cationic lipid transfectam (Behr, et al., 1989), if there is a small positive charge excess (1.5 and 2 charge equivalents), d. H. at suboptimal Concentration, is used by means of membrane-active acidic peptides can be significantly enhanced.

The enhancement was INF6 in the case of the peptide (2 µg / 1.5 charge equivalents of Transfectam) up to 1000 times what the level of with optimal amounts Transfectam (high positive charge excess) achieved gene expression. The reinforcement gene expression with membrane-active peptides was observed several cell types. Among the tested membrane-active peptides only brought the acidic good ones Results in combination with Transfectam. This Findings are in accordance 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 what the lipid / DNA complexes is guaranteed if the peptides are ionic to the Transfection particles are bound. When using 2 charge equivalents of Transfectam was the order the performance of the peptides INF6 <INF10 < EGLA-IINF5 <INFA <Melittin. This for the examined Peptides obtained with Transfectam give way based on findings obtained when the peptides in the transfection system based on the receptor mediated endocytosis as part of Transfection complexes containing transferrin-poly lysine: INF5 was used in this system the best peptide and about 50 times more effective than INF6, while in combination with Transfectam 10 times is less effective than INF6. The INF5 peptide has one membrane-breaking effect only at acidic pH, while INF6 membrane active even at neutral pH is. Based in the lipid-free gene transfer system of polylysine-conjugated cellular ligands was the Activity of the peptide at a neutral pH of accompanied toxic side effects that were not observed were when the peptide was used in the lipid system were. The artificial peptide EGLA-I and INF10 were shown to be an additive to lipid / DNA complexes more suitable than in polylysine-containing complexes.

It was also found that the effect of tested peptides on the transfection efficiency of cationic lipids with suboptimal charge ratio was of the same order of magnitude as the effect of Helper lipids (Remy, et al., 1995; Zhou, et al., 1994; Felgner, et al., 1994; Leventis, et al., 1990).

The transfection complexes containing cationic Lipid and membrane-active peptide, according to different methods can be produced. In the  Attempts of the present invention have been made Compositions according to three different methods made, with the methods in order the addition or combination of the complex components and the time of dilution distinguished. In the first variant, the DNA after combining peptide with transfectam admitted; in the second variant was first Transfectam complexed with DNA and then that Peptide added. In the third variant that was Peptide after dilution of the transfectam / DNA complex admitted. It was examined whether the type of Production of the transfection complexes have an impact the extent of the increase in transfection efficiency Has. It turned out that the Manufacturing method on the different peptides affects differently: For INF6, the three manufacturing methods as equivalent. With INF5 brought the by the second and third method manufactured transfection complexes better results, while with INF10 the first and third variant of the second were superior.

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 (Remy, et al., 1995) at low charge (2 charge equivalents transfectam) were compared with the complexes containing a membrane-active peptide of the present invention with regard to 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.

It was also found that the membrane active Peptide the sensitivity of the transfection complexes decreased compared to serum, which in terms of Application in vivo is important.

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 shows effect of different membrane-active peptides on transfection;

Fig. 5 Effect of preparation method of membrane-active peptide containing transfection to transfection;

Fig. 6 efficiency of membrane-active peptide-containing complexes when transfected various cell lines;

Fig. 7 Effect of bafilomycin A1 on transfection;

Fig. 8 influence of helper lipids on the transfection efficiency;

Fig. 9 Influence of the combination of Transfectam, helper lipid and membrane-active peptide on transfection;

Fig. 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 (M stands for mol l ^-1 ):

a) Reporter gene plasmid pCMV-Luc

The construction of the plasmid pCMV-Luc that the Luciferase gene under the control of the  CMV promoter / enhancer has been named pCMVL is described in WO 93/07283 A1.

b) Various reagents

The lipopolyamine DOGS (dioctadecylamidoglyclyspermin) with the trade name "Transfectam" was from Promega related, the helper lipids DOPE (1,2-dioleoyl-sn-glycero-3- phosphoethanolamine), MOG (1-mono-oleoyl-rac-glycerol), DOG (1,2-di-oleoyl-rac-glycerol), EPE (Egg phosphatidylethanolamine), EPC (egg phosphatidylcholine), as well as chloroquine, bafilomycin A₁ and melittin (from Bee venom) were obtained from Sigma.

c) peptide synthesis

The name of the peptides, their origin as well Sequences are given in the table.

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

The INFA, INF10 and EGLA-I peptides have been identified under Using the Fmoc strategy (N- (9- Fluorenyl) methoxycarbonyl) with HBTU activation [O- (1H-benzotriazol-1-yl) -N, N, N ′, N′- tetramethyluronium hexafluorophosphate], (Fastmoc® - 0.25 mmol scale) on an Applied Biosystems Model 433 A peptide synthesizer (Foster City, California) with feedback monitoring. Per Cycle were three deprotection steps ("Deprotection") carried out. If the feedback mon nitoring revealed that Fmoc deprotection was not was quantitative, the next step was a Double coupling of the next amino acid and blocking  the terminal NH₂ groups (by Acetic anhydride). It was the following amino acid protecting groups used: (Trt) Asn, (Trt) Cys, (t-Bu) Cys, (t-Bu) Asp, (t-Bu) Glu, (Boc) Lys. A mixture of 70% NMP / 30% DMF used.

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 for the synthesis of EGLA-I used.

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

The peptide called INF7dimer (a dimer of the peptide INF7 described by Plank et al., 1994) was based on the Fmoc strategy Applied Biosystems Peptide Synthesizer Model 431 synthesized. It became a preloaded with cysteine Resin used (Tentagel S PHB-Cys). Be the first Amino acid became the medium-sized Lys DiFmoc-ge couples. Thereafter, up to Ile-18 were duplicated carried out. From Met-17 to Ile-10 Single pairings performed; at Phe-9 one Single coupling, then additional coupling with 1% Triton in the coupling approach. From Gly-8, simple ones became Pairings performed.

The deprotection of the peptides and the cleavage of the Resin were mixed with a mixture of phenol, Ethanedithiol, thioanisole, water and trifluoroacetic acid  (0.75: 0.25: 0.5: 0.5: 10). The raw peptides were precipitated by dropwise addition to ether and centrifuged. The peptides thus obtained were washed three times with ether and then under an argon stream followed by high vacuum. The crude peptides were in 1 M TEAB, pH 9 and 1% β-mercaptoethanol dissolved.

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

The EGLA-I peptide underwent gel filtration (Sephadex G10, 20 mM TEAA, pH 7.3). The purified peptide fraction was in a Speedvac (Savant) freeze-dried. The analytical Reverse phase chromatography showed a purity of about 95%.

The solutions of the raw peptides INF10 and INFA were by means of gel filtration on Sephadex G10 (10 mm × 300 mm Column) fractionated in 20 mM TEAA, pH 7.3. The peptides were lyophilized; the analytical reverse phase chro matography showed a purity of approx. 98 or 95%.  

The INF7dimer peptide was purified on Sephadex G 10 (Buffer HBS; column 10 mm × 300 mm). Then one became Ion exchange chromatography carried out (column: (10 mm x 100 mm); Material: Toso Haas Super Q 650 S; Flow rate: 0.5 ml / min; Eluens: 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, i.e. H. 1% / min). The peptide eluted at 70-80 min. That cleaned over ion exchangers The peptide was again passed through a Sephadex G 10 column (10 mm × 300 mm) cleaned (buffer HBS / 50% glycerin).

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

The Liposome Permeability Assay ("Liposome Leakage Assay ") was performed as described by Plank, et al., 1994.

d) cell culture and media

The media as well as fetal calf serum (FCS) and Horse serum was purchased from Gibco-BRL. The Culture media were mixed with 2 mM L-glutamine and antibiotics added. Primary human melanoma cells were used as cells the designation H225, mouse embryo liver cells of the Cell line TIB-73 (ATCC BNL CL.2), human Lung carcinoma cells of cell line A549 (ATCC CCL 185) and cells of the mouse melanoma cell line Cloudman 591, clone M3 (ATCC No. CCL 53.1) used. The H225 cells were in RPMI 1640/10% FCS / 1 mM sodium pyruvate, which 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 made as by Barthel, et al., 1993, wherein 3 µg of plasmid DNA and the amount of Transfectam used in each 75 ul 150 mM NaCl were dissolved. After 10 to 20 min the two solutions were combined. After another The mixture was exposed to serum-free medium for 10 min diluted a total volume of 2 ml.

The expression "charge equivalent" indicates the amount cationic lipid for transfection was used; 1 charge equivalent corresponds to the Amount that is required to remove all negative charges neutralize the phosphate groups of the plasmid.

3 µg DNA corresponds to 9 nmol negative charges; in the Calculation of the charge ratio was takes into account that 1 mole of transfectam is 3 moles positive Has charges that of the three in physiological protonated ammonium groups. Outgoing with a molecular weight of approx. 1250 it means that at Use 3 µg DNA for a double positive Charge excess (2 charge equivalents Transfectam / 3 µg DNA) 3 µl of a 2 mM transfectam solution, containing 6 nmol (= 7.58 µg) transfectam required.

The peptides were used in an amount of 0.5 or 1 mg / ml Solution in HBS (containing 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 with culture medium to 2 ml brought from the transfection mixture obtained 1 ml was pipetted onto the cells.  

The helper lipids DOPE, DOG, EPC, EPE or MOG or Cholesterol was in ethanol containing a trace Dichloromethane, diluted. The Transfectam / helper lipid / DNA complexes were formed by changing the respective amounts of transfectam / helper lipid (the amount of helper lipid used, based on Transfectam, is given as a molar ratio) before Dilution were mixed with the DNA solution.

ii) transfection and luciferase determination

50,000 to 75,000 cells per well of a 24 well plate were a day before transfection plated out, in all experiments the Transfection volume was 1 ml. The transfection was with 1 ml of the complex prepared in i) carried out. After 3 to 4 hours that was Transfection medium through fresh medium containing 10% FCS, replaced. Every experiment was, unless it was otherwise stated, carried out at least twice, where the indicated in the pictures Peptide levels in these cases total for that Obtain duplicate amount used.

The cells were harvested 24 hours after the transfection and in 150-200 µl 250 mM Tris pH 7.3, 0.5% Triton X-100 added. The cell lysate was then in Transfer 1.5 ml Eppendor tubes and add 5 min 14,000 g centrifuged to remove the cell debris pelletize. Determination of luciferase activity was carried out as described in WO 93/7283, the luciferase light units from an aliquot of the supernatant (20 µl) with 10 sec integration after Injection of freshly prepared luciferin solution was determined. The luciferase background (150-250 Light units) was subtracted from each value; the  Transfection efficiency was expressed as Total light units, which are the mean of Represent duplicate determinations. For quantitative The Bradford assay was used to determine the protein content (Bio-Rad) used.

f) flow cytometry

The plasmid DNA was used for the flow cytometry 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) incubated, then the complexes were prepared as described above. The complexes were made up of 300,000 H225 cells per Was given a well of a 6 well plate, then was 4 h long either at 4 ° C (cell surface association) or at 37 ° C (cell surface association and absorption in the cells) incubated. After that, the cells were made twice washed with cold PBS and with 1 mM EDTA in PBS harvested and on a FACScan device (Becton Dickinson) analyzed.

example 1 Transfection efficiency of Transfectam / 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 tests is shown in Fig. 1 (dotted line with rhombuses: 1 equivalent (eq.) Transfectam / x µg INF6; full line with circles: 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 1000-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 Figure 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, high luciferase expression is 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) or 20% (gray bars) of FCS which was 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 of transfectam did not result in a significant change in the curve profile at either 4 ° C or 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. was measured with various transfection complexes (the cells were incubated at 4 ° C. or at 37 ° C. after 4 h washed twice with PBS, mixed with fresh medium containing 10% FCS and incubated for 20 h at 37 ° C and 5% CO₂). The presence of peptide (1.5 µg / well; a simple determination was carried out in this experiment) resulted in a 15-fold increase in expression when the mixture was 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, transfectam / 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 made in three different ways ( Fig. 5):

• a) Association of peptide and lipid before Complexation with DNA (dotted bars)
• b) adding the peptide after formation of the transfectam / DNA com plexes 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. The combination of the peptide with Transfectam before complexation with the plasmid resulted in a contrary behavior of the peptides: while the gene expression rate remained unchanged at INF6, the performance of the complexes produced at INF10 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 peptides on transfection efficiency 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 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 peptide.

Example 7 Examination of the influence of bafilomycin A₁ on the Transfection efficiency of transfectam / DNA complexes

The presence of the specific inhibitor of the vacuole proton pump bafilomycin A 1 (Bowman, et al., 1988; Yoshimori, et al., 1991) in 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 were used; after 4 h of incubation, the medium was replaced by fresh one with a content of 10% FCS), gene expression decreased by a factor of 7, 5 or 1.5 ( Fig. 7 Empty bars: without adding Bafilomycin A1.Dotted bars: in the presence of 200 nM bafilomycin A1). 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) Strengthening the effect of Transfectam by 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 10 to 20 times higher gene expression (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 reinforcement was achieved if the transfectam / DOPE formulation is 1 mol /% DOG was added; this is probably due to the  Ability of DOG attributed u. a. a merger too induce (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 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 of the cationic lipids alone ( Fig. 3B). When the experiment was carried out at 37 ° C, the differences in luciferase expression were 2 and 640 times, respectively.

b) Strengthening 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 gave no further increase in complexes with high efficiency (Transfectam 2 eq./INF10 3 µg, 5 µg or 7.5 µg / 1.5 eq. DOPE) (not shown in FIG. 9). However, when the peptide was used with an excess of transfectam (6 charge equivalents), a significant increase in gene expression could be obtained over the values obtained with optimal amounts of transfectam / DOPE; 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 called INF7dimer (see table) Dimeres of that described by Plank, et al., 1994 Peptide INF7, was linked to the lipid derivative DPPE- (Dipalmitoylphosphatidylethanolamyl) bromoacetamide (Hexadecanoic acid-3 - ((2- (2- (2- (2- (2- (2- Bromoacethylamino) ethoxy) ethoxy) ethoxy) acetylamino) ethoxy) hydroxyphosphoryloxy) -2- hexadecanoyloxypropyl ester) coupled by 0.95 equivalents of peptide (80 nmol; approx. 450 µg) to that in 1.3 ml diethanolamine buffer (pH 9.5 / ethanol; 9/1; v / v) dilute lipid were added. After 3 hours at At room temperature, an Ellman test was quantitative Determination of the thiol residues carried out. Subsequently was used to destroy the rest Bromoacetamide group β-mercaptoethanol added. The  Lipid-modified peptide obtained was without further Cleaning used.

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 up 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 (28)

1. Composition for the transfection of higher eukaryotic cells, said composition comprising a complex containing one, in the cell to be expressed nucleic acid, as well as, in a sub-optimal for transfection concentration, one or more cationic lipids, and optionally containing helper lipid (s), characterized 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 0 to 3. 2. Composition according to claim 1, characterized characterized that the ratio of positive to the negative charges of the composition 0 to 2. 3. Composition according to claim 1, characterized characterized that the suboptimal concentration of cationic lipid, optionally in a mixture with helper lipid, corresponds to a concentration, where the transfection efficiency is a factor of at least 2 is less than the optimal one Concentration. 4. Composition according to one of claims 1 to 3, characterized in that it is a peptide of Designation INF6 with the sequence GLF GAI AGFI ENGW EGMT DGWYG contains.   5. Composition according to one of claims 1 to 3, characterized in that it is a peptide of Designation INF10 with the sequence GLF ELA EGLA ELGW EGLA EGWYGC contains. 6. Composition according to one of claims 1 to 3, characterized in that it is a peptide of Designation INF5 with the sequence [GLF EAI EGFI ENGW EGnIDG] ₂ K contains. 7. Composition according to one of claims 1 to 3, characterized in that it contains the peptide Designation EGLA-I with the sequence GLFL GLA EGLA EGLA EGLA EGLA EGLEGLAGGSC contains. 8. Composition according to one of claims 1 to 3, characterized in that it is a peptide of Designation INFA with the sequence GLF EAI EAFI ENAW EAMI DAWYG contains. 9. Composition according to one of claims 1 to 3, characterized in that it is a peptide of Designation INF8 with the sequence [GLF EAI EGFI ENGF EGMI DGGG] ₂ K contains. 10. Composition according to one of claims 1 to 3, characterized in that it is a peptide of Designation INF9 with the sequence GLF ELA EGLA ELGA EGLA contains EGWYGC. 11. The composition according to any one of claims 1 to 3, characterized in that it is a peptide of Name EGLA-II with the sequence WEA GLA EGLA EGLA EGLA EGLA EGL EGLA GGSC contains.   12. Composition according to one of claims 1 to 3, characterized in that it is a peptide of Designation EGLA-III with the sequence GLF EGA EGLA EGA EGLA EGLA EGWY GAC contains. 13. Composition according to one of claims 1 to 3, characterized in that it is a peptide of Designation EGLA-IV with the sequence GLF EGA EGLA EGW EGLA EGLA EGWY GAC contains. 14. Composition according to one of claims 1 to 3, characterized in that it is a peptide of Name INF7dimer with the sequence [GLF EAI EGFI ENGW EGMI DGWYG] ₂ KC contains. 15. Composition according to one of the preceding Claims, characterized in that the membrane active peptide modified with a lipid is. 16. The composition according to claim 15, characterized characterized in that the peptide with Dipalmitoylphosphatidylethanolamyl (DPPE) is modified. 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 characterized in that the lipopolyamine Dioctadecylamidoglyclyspermin (DOGS) is. 19. Composition according to one of the preceding Claims, characterized in that they are a or more helper lipids selected from the  Group phosphatidylethanolamines, Phosphatidylglycerols and diacylglycerols, contains. 20. The composition according to claim 19, characterized characterized as a helper lipid Contains dioleylphosphatidylethanolamine (DOPE). 21. The composition according to claim 19, characterized characterized in that they serve as helper lipid oleoyl contains palmitoylphosphatidylethanolamine (POPE). 22. The composition according to claim 19, characterized characterized that they are helper lipid 1-mono oleoyl-rac-glycerin (MOG) contains. 23. The composition according to claim 19, characterized characterized that they are helper lipid 1.2-di- Contains oleoyl-rac-glycerin (DOG). 24. The composition according to claim 19, characterized characterized as a helper lipid Contains egg phosphatidylcholine (EPC). 25. The composition according to claim 19, characterized characterized as a helper lipid Contains egg phosphatidylethanolamine (EPE). 26. Pharmaceutical preparation containing as active component according to a composition one of claims 1 to 25. 27. Process for transfection of higher eukaryotic Cells, characterized in that the cells with a composition according to any one of the claims 1 to 25 in contact.   28. Application of the method according to claim 27 Mammalian cells in vitro or ex vivo.