DE19925143A1 - New liposomal vector complexes and their use for gene therapy - Google Patents

Abstract

The invention relates to a novel liposomal vector complex, containing the following components: a) a nucleic acid sequence of any length; b) a cationic support which condenses the component a) and is lysosomolytic and/or lysosomotropic; c) lipids and phospholipids which form a liposome; d) optionally a ligand which has a connecting point for a target cell; and e) optionally a fusogenic substance which can replace the lysosomolytic and/or lysosomotropic functions of component b). If a fusogenic substance (e) is present, the cationic support (b) need not be lysosomolytic and/or lysosomotropic. The invention also relates to the production and use of said complex.

Description

So far, the essential components of vectors for gene therapy are Nucleic acid sequences which are linked to a non-viral carrier (e.g. cationic Lipids or cationic polymers) complexed or inserted into a virus.

Previous experience with such vectors in gene therapy various diseases, especially tumor diseases, shows that especially non-viral vector complexes are only relative in cell culture can transduce small numbers of cells (mostly between 1% and 30%); of further that after administration of a gene therapy vector in the Circulation of an organism these vectors in a short time from the circulation are eliminated and for binding to the target cells and for transfection of these Target cells are no longer available (Ogris et al. Gene Ther. 6: 595, 1999; Dash et al., Gene Ther. 6: 643, 1999; Li et al., Gene Ther. 5: 930, 1998; Liu et al. Gene Ther. 4: 517, 1997).

This elimination can be done by degradation of the DNA or by rapid Deposition of the vectors in the lungs, liver or the so-called reticuloendothelial system (RES), which is specially developed in the spleen and the lymph nodes (Zhu et al., Science 261: 209, 1993).

The causes of rapid elimination are varied. They can be: one too large negative or positive charge, too large a volume or opsonization the vector particles through blood proteins. With viral vectors, they can also be the binding of the virus envelope proteins to virus-specific receptors in the Organs and / or antibodies or immune cells specific for the viruses which bind to the vectors and thereby eliminate them.  

Previous experience also shows that the coupling or insertion of a target cell-specific ligands in the vector complex whose rapid elimination not significantly reduced after administration to the bloodstream.

Knowing these problems, there is an urgent need for new ones Preparations of vectors which transfect as many cells as possible in the cell culture and which after administration in a living organism for as long as possible Cycle remain and cannot be eliminated prematurely from the cycle. To the Elimination of cationic lipids or cationic polymers in complex with To reduce nucleic acid sequences from the bloodstream has been polyethylene glycol (Senior et al., Biochim. Biophys. Res. Acta 1062: 77, 1991; Mori et al., FEBS Lett 284: 263, 1991; Ogris et al., Gene Ther. 6: 595, 1999), vinyl polymers (torchiline et al., Biochim. Biophys. Res. Acta 1195: 181, 1994) or other amphipathic Polymers (Woodle et al., Bioconjugat. Chem. 5: 493, 1994) to the cationic Lipids or cationic polymers coupled or with the help of negatively charged lipids anionic liposomes, in which the nucleic acid sequences in the Includes complex with cationic lipids or cationic polymers goods (U.S. Patent No. 4,946,787; U.S. Patent No. 4,245,737; U.S. Patent No. 5,480,463; Heywood and Eanes, Calc. Tissue int. 40: 149, 1992; Lee and Huang, J. Biol. Chem. 271: 8481, 1996; Balicki and Beutler, Blood 88: 3884, 1996; Lucie et al., J. Lip. Res. 8: 57, 1998; Lakkaraju et al., J. Lip. Res. 8: 74, 1998; Turner et al., J. Lip. Res. 8: 114, 1998; Schoen et al., J. Lip. Res. 8: 485, 1998).

Such modifications led to a stabilization of the Vector particle size, inhibited the aggregation of the vectors with themselves or with Blood cells reduced the opsonization of vectors by binding Protect immunoglobulins, complement factors, fibrinogen or fibronectin (adeno) viral vectors before elimination by antibodies (Chillon et al., Gene Ther. 5: 995, 1998) and caused the blood residence time to be increased from Vectors, a significantly stronger accumulation in subcutaneously growing tumors and a transduction of the tumor cells (Ogris et al., Gene Ther. 6: 595, 1999).  

At the same time, however, a considerable amount could also be found in the lungs, spleen and liver Enrichment of the vectors and transduction of the tissue cells in these organs are found (Ogris et al., Gene Ther. 6: 595, 1999) so that it can be concluded may, for example, the coupling of PEG is an improvement, but still does not optimize the distribution of vectors.

General description of the invention

The invention relates to new üposomal vector complexes for gene therapy consisting of the following components:

• a) a nucleic acid sequence of any length;
• b) a cationic carrier which condenses component a) and at the same time has lysosomotropic properties; and
• c) Lipids and phospholipids, which form a liposome.

Component a) can be an unmodified or modified DNA sequence or be an unmodified or modified RNA sequence. The nucleotide sequence can have an anti-DNA (triplex) or anti-RNA (antisense; ribozyme) function or code for an RNA sequence acting in this way or for a protein. The Nucleotide sequences and their modification can be such that the Nucleotide sequence is largely resistant to degradation by DNAsen or RNase. Examples of such nucleotide sequences and their modifications are shown in Breaker, Nature Biotechnol. 15: 427, 1997; Gerwik, Critical Reviews in Oncogenesis 8: 93, 1997; Mukhopadhyay et al., Crit. Rev. Oncogen. 7: 151, 1996; Mercola et al., Cancer Gene Ther. 2: 47, 1995; Frank-Kamenetski, Annu. Rev. Biochem. 64: 65, 1995 and Fraser et al., Exp. Opin. Invest. Drugs 4: 637, 1995. The DNA Sequence can be linear or circular, for example in the form of a plasmid.

Component a) can also be a virus, preferably a virus, in which with the methods known to those skilled in the art is foreign to the virus Nucleic acid sequence was inserted. Examples of such viruses are RTV, AAV  and lentiviruses. Such and further examples are from Vile, Nature Biotechnol. 15: 840, 1997; McKeon et al., Human Gene Ther. 7: 1615, 1996; Flotte et al., Gene Ther. 2: 357, 1995; Jolly, Cancer Gene Ther. 1:51, 1994; Dubensky et al., J. Virol. 70: 508, 1996.

Component b) is a cationic carrier which contains component a) condensed and at the same time has strong lysosomotropic properties.

According to this invention, in a particular embodiment, the Component b) is a cationic polymer, for example described by Boussif et al., Proc. Natl. Acad. Sci. USA 92: 7297, 1995; Kaneda et al., Science 243: 375, 1989; Keown et al., Methods in Enzymology 185: 527, 1990; Baker et al., Gene Ther. 4: 773, 1997; Fritz et al., Human Gene Ther. 7: 1395, 1996; Wolfert et al., Human Gene Ther. 7: 2123, 1996 and Solodin et al., Biochem. 34: 13537, 1995.

In a further particular embodiment of this invention, the Component b) is a polyethyleneimine (PEI), in another special one In one embodiment of this invention, the polyethyleneimine has a molecular weight in a range of 500-10,000 Da and in another embodiment Molecular weight averaging about 2000 Da and was made as in patent application EP-A 0 905 254.

Component c) represents any liposome with any, the expert known composition. In a particular embodiment, this Liposome has an anionic charge. In a further special embodiment the lipid and phospholipid composition of the anionic liposome resembles that Composition of a virus envelope. The production of liposomes with anionic Cargo has been described many times, for example in the US Patents No. US 4,946,787, US 4,245,737, US 5,480,463, and Heywood and Eanes, Calc. Tissue int. 40: 149, 1992; Lee and Huang, J. Biol. Chem. 271: 8481, 1996; Balicki and Beutler, Blood 88: 3884, 1996; Lucie et al., J. Lip. Res. 8: 57, 1998;  Lakkaraju et al., J. Lip. Res. 8: 74, 1998; Turner et al., J. Lip. Res. 8: 114, 1998; Schoen et al., J. Lip. Res. 8: 485, 1998.

For example, the preparation of liposomes that are similar to virus envelopes has been demonstrated described in U.S. Patent Nos. 5,252,348; US 5,753,258; US 5,766,625 and EP-A 0 555 333.

The invention furthermore relates to the addition of the invention liposomal vector complexes by adding a component d).

This component d) represents a ligand which is a binding site for the Target cell and is conjugated to a lipid. The target cell specificity of the ligand can be any.

Target cell specificities of ligands selected from a group are preferred described in detail in

• - Multi-functional ligand systems for the target cell-specific transfer of Nucleotide sequences EP-A 0 846 772
• - Single-chain, double-antigen-binding molecules (DE 1 981 61 417, yet unpublished)
• - specifically cell membrane penetrating molecules (DE 198 50 987.1, yet unpublished) or
• - Target cell-specific, multivalent proteins (DE 199 10 419.0, still unpublished).

The type of lipid can be of any type, but is of course preferred occurring lipids, such as, for example, in US Pat. No. 5,252,348; US 5,753,258; US 5,766,625 and EP-A 0 555 333.

The lipid is conjugated to the target cell-specific ligand using one of the Methods known to the person skilled in the art, for example as in US Pat. No. 5,662,930  described.

The insertion of component d) into the liposome according to the invention (component c) is carried out using the method known to the person skilled in the art, for example described in U.S. Patent Nos. US 5,252,348 and US 5,753,258).

The invention furthermore relates to the addition of the invention liposomal vector complexes by adding a component e). This Component e) preferably represents the functional sequence of a fusion peptide the subunit HA-2 of hemagglutinin from the influenza virus (Wagner et al., Proc. Natl. Acad. Sci. USA 89 (17), 7934-7938) ligands which inhibit the release of the Liposome content relieved from the endosome.

The vector complex according to the invention consisting of components a), b) and c) or a), b), c) and d) or a), b), c), d) and e) is prepared using those known to the person skilled in the art Methods, such as that

• - In step 1 component a) is mixed with component b) preferably in the molar ratio [a): b)] of 1: 1 to 1: 1000, the Mixing ratio should be set so that the net charge of the resulting overall complex preferably either cationically or is anionic and subsequent
• - in step 2, the complex resulting from step 1) into component c) is introduced and
• - in a third step component d) is inserted into component c).
• - in a 4th step component e) in the resulting from step 2 or 3 Complex is inserted.

The liposomes resulting from these manufacturing steps are preferred a diameter of 100-300 nm and an anionic charge.

Such liposomal vector complexes according to the invention are characterized by their Component c) largely shielded, d. H. their binding and transfection and Transduction of cells that do not have a receptor for component d) within the Carrying vector complex according to the invention is largely reduced.

As a result, after administration of the liposomal invention Vector complexes in an organism, either locally, on the skin, in an organ, a body cavity, into a connective or supportive tissue or into the bloodstream, the Residence time significantly extended after administration to the bloodstream for example up to several hours to a few days.

Over this long dwell time, those of the invention accumulate Liposomes, for example, in the tumor vessel bed due to the so-called "passive targeting" (Unezaki et al., Int. J. Pharmaceutics 114: 11, 1996; Sadzuka et al., Cancer Lett. 127: 99, 1998 and Wunder et al., Int. J. Oncol. 11: 497, 1997). Furthermore, and regardless of passive targeting, the Liposomes according to the invention via their component d) to the target cell and transfect them so that the nucleic acid sequence in the invention Vector complex is released in the cell.

Depending on the composition, this nucleic acid sequence can be in the target cell Develop effect, d. H. for example the transcription or translation of a inhibit certain genes or a certain RNA, or the cell transduce to the expression of the RNA or the protein encoded by this Nucleic acid sequence.

The transduction rate through the liposomal vector complexes according to the invention is compared to the existing technology known to the person skilled in the art improves and lies, for example in cell culture, in over 80% of the cells which  carry a receptor for component d) and with the invention liposomal vector complexes are brought into contact.

The vector complexes according to the invention are therefore preferably suitable for the in vitro transduction of cells and for in vivo administration with the aim of Prophylaxis or therapy of diseases.

Examples to illustrate the idea of the invention

The following examples are intended to illustrate how to make the present invention could execute.

example 1 Production of a liposomal vector complex according to the invention Production of component a)

The plasmid "pEGFP-C1" from Clontech was used as component a) and contains the following nucleotide sequences:

• - CMV promoter
• - cDNA for the "enhanced green fluorescent protein" (EGFP)
• - SV40 poly A signal

Using the methods known to the person skilled in the art, the plasmid in E. coli bacteria introduced, the bacteria multiplied in culture medium and the plasmids isolated.

Production of component b)

Low molecular weight PEI-2000 was made as in the patent application EP-A 0 905 254. For this purpose, a 10% ethyleneimine monomer Solution in water (5 ml ethyleneimine monomer + 45 ml distilled water, dissolution with stirring) with the addition of 1% (0.5 ml) concentrated hydrochloric acid (37 ° C) as  The catalyst was stirred for 4 days at 50 ° C., evaporated and under vacuum Room temperature dried. The molecular weight determinations were carried out using Laser scattered light measurement (Wyatt Dwan DSP light scattering photometer) at 633 nm Direct injection into a K5 measuring cell. The molecular weights are based on the calibration constants determined in toluene and the known sample weight certainly.

Molecular weight determination using light scattering analysis gave 2000 Da. By comparison, the PEI obtained commercially (from Fluka, Neu Ulm) had one Molecular weight according to the light scattering analysis of 791 kDa (HMW-PEI).

Production of components c) and d)

Synthesis of the DPPE derivative (anchor lipid)

material

DPPE is dissolved in freshly distilled, anhydrous chloroform (1 week over Mg sulfate; 2 days over phosphorus pentoxide). Glutaric anhydride and anhydrous pyridine are added to the mixture and the mixture is stirred at 20 ° C. for 2 days. The detection of the desired reaction product is carried out by means of thin layer chromatography (TLC), followed by the narrowing of the batch to dryness by means of a vacuum pump. The mixture is taken up in chloroform and the N-glut-PE is isolated by means of a preparative TLC (Merck, silica gel plates 60 F 254) and dissolved out of the silica gel with methanol. After removal of the methanol, the ready-to-use N-glut-PE stock solution (2 µmol / ml) is obtained by dissolving the N-glut-PE in chloroform. The product was detected by ¹ H NMR [2].

literature

[1] Volker Weissig, habilitation thesis in physiological chemistry, 1991, 28-29
[2] Ahl et al., (1997), Biochimia et Biophysica Acta 1329: 370-382.

Modified peptide with RGD sequence

A cyclic was used to improve the targeting of the integrin receptors Peptide synthesized by IMT (here input Dr. Krause). It's about a CDCRGDCFC peptide (SEQ ID NO: 1) with an additional arginine at the N- terminal end (FW 1163.35). The terminal amino acid reduces the extent the coupling of the peptide to the active center, the RGD sequence.  

The cyclization takes place by oxidation of the thiol groups to disulfide bridges. The successful cyclization is checked by means of HPLC analysis.

After HPLC purification, the peptide is lyophilized, stored at 4 ° C and dissolved in buffer before use (250 µg / 150 µl Tris buffer 10 mM pH 7.4 or PBS- Buffer pH 7.4).

RGD with additional arginine at the N-terminal C, FW: 1163.35

Production of liposomes [1]

material

Stock solutions

The liposomes are produced using the film / hydration method. In the Chloroform dissolved lipids and the lipid anchor are placed in a 100 ml round bottom flask pipetted and the chloroform removed for 15 min. The piston plunges into it temperature controlled water bath with a temperature above the The phase transition temperature of the lipids is 30 ° C in this special case. For complete removal of the solvent, the film is 15 min under high vacuum dried.

The film is then hydrated with buffer (Tris 10 mM, pH 7.4 or PBS pH 7.4). The buffer is added to the flask together with a few small glass beads and the batch is rolled for 45 min under N ₂ gassing. Here too, the flask is immersed in the 30 ° C water bath. To swell the lipid film and to produce multilamellar liposomes (MLV), the mixture is left to stand for 2 hours at room temperature [1, p. 38]. The MLV suspension is transferred to a special Sonicator glass vessel and the batch is sonicated for 15 seconds using a Probe Sonicator (here Soniprep 150 (preferred amplitude in microns 8-12)). The suspension is immersed in an ice bath [1, pp. 44-47]. After sonication, a pause of 30 seconds is taken to cool the suspension. This procedure (sonication - pause) is repeated 10 times. The size measurement of the SUV obtained results in a size of 120 to 300 nm. After subsequent extrusion [1, pp. 52-56] [2] using LiposoFast through a polycarbonate filter, pore size 50 nm, the size in this example is reduced to a value between 107.5-128 nm. The liposomes thus produced are stable for at least 2 months and during this time their size cannot be measured. The liposomes are filled into a sterile Eppendorf cone under the LF for further processing.

literature

[1] Roger RC New, "Preparation of liposomes", chapter 2, Liposomes a practical approach (1989)
[2] Olson et al. (1980) Biochim. Biophys. Acta, 394, 483

Coupling the peptide to lipid anchors [1]

material

First the carboxyl group of the glutaric acid residue of the lipid anchor is activated by the addition of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide [3, p. 170]. For this, 400 µl of the liposome suspension (4 µmol total lipid, 0.4 µmol N-glut-PE, medium Tris buffer pH 8, 10 mM) are vortexed and 3.5 mg EDC are added. The approach is vortexed again and protected from light for 5 h, shaken (IKA-Vibrax-VXR). The activated intermediate, the O-acyl intermediate, to which the peptide with a free amino function binds to form an amide, is formed. 250 µg RGD dissolved in PBS buffer pH 7.4 or Tris buffer 10 mM pH 7.4 (250 µg / 150 µl) are added, the mixture is vortexed and left to shake overnight. After coupling, the unbound peptide is separated from the liposomes by gel chromatography. The size exclusion chromatography is carried out with a Sephadex G 50 column, the eluent is Tris buffer 10 mM, pH 7.4.

1. Activation with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide

2. Coupling of the peptide to the amino group

The coupling yield is measured using a simultaneous approach Fluorescent dye (5-DTAF) labeled RGD performed [4] and lies at least at 6 µg RGD / 1 µmol PL (calculated for the actual amount of lipid with Cholesterol). The size of the liposomes coupled with RGD is between 100-150 nm.

literature

[1] Weissig, V., Lasch, J., Klibanov, AL Torchilin, VP, A new hydrophobic anchor for the attachment of proteins to liposomal membranes. FEBS Lett. 202, 1986, 86-90
[2] Diploma thesis Ragna Schmidt, University of Halle (1997)
[3] Martin et al. "Covalent attachment of protein to liposomes", Chapter 4, Roger RC New, Liposomes a practical approach (1989)
[4] Product Information Sheet 5-DTAF, Molecular Probes (MP 00143 08/19/98) "Conjugation with Amine-Reactive Probes"

Complex formation

material

Approach for a 3 cm cell culture dish

The complex is first formed by combining the negatively charged ones Components of plasmid DNA and liposomes. Thereby is the dilution of the solutions to be observed to prevent irreversible precipitate formation. The final volume of all constituents mentioned is 100 µi. First the buffer (Tris 10 mM, pH 7.4, other buffers also possible) and 10 µg plasmid (10 µg / 60 µl) and 40 µg Liposomes (variable, between 3 and 6 µg / µl) combined by easy pipetting. The mixture is vortexed.

The DNA is then condensed by adding the cationic agent, first add 19.96 µg protamine sulfate (charge ratio +/- 3.3). To the protamine sulfate is quickly added with an Eppendorf pipette and the Mixing by pipetting up and down 10 times and coating the Complex achieved with the lipids. Then add 29.7 µg (quantities up to 17.93 µg are also possible) PEI (charge ratio +/- 20.7, reduction  up to 12.5), another cationic agent. For this, 33 µl PEI solution 0.9 mg / ml diluted with 250 µl ultrapure water and prepared in increments admit. The addition takes place in 2 * 100 µl and 1 * 85 µl steps, each 10 * the batch pipette up and down after the addition, wait 15 min. Such a complex has 1 h after producing a size of 180-230 nm and will immediately after Manufacture used for cell culture experiments. The complexes are in the for the transfection used cell culture medium M199 + 10% FCS stable (size 360-400 nm).

The batch can also be prepared without protamine sulfate, Transfection experiments show almost identical efficiency.

Approach for a 3 cm cell culture dish

The complex is first formed by combining the negatively charged ones Components of plasmid DNA and liposomes. Thereby is the dilution of the solutions to be observed to prevent irreversible precipitate formation. The final volume of all ingredients mentioned is 100 ul. First the buffer (Tris 10 mM, pH 7.4, other buffers also possible) and 10 µg plasmid (10 µg / 60 µl) and 40 µg  Liposomes (variable, between 3 and 6 µg / µl) combined by easy pipetting. The mixture is vortexed. The condensation then takes place the DNA by adding the cationic agent, from 29.7 µg (amounts up to 17.93 µg are also possible) PEI (charge ratio +/- 20.7, reduction to 12.5 possible). For this purpose, 33 µl PEI solution 0.9 mg / ml are diluted with 250 µl ultrapure water and add in increments to the approach. The addition is made in 2 * 100 µl and 1 * 85 µl Steps, each 10 * the batch after addition and pipette off, wait 15 min. Even simply by mixing all of the specified components into complexes achieved similar effectiveness.

Such a complex has a size of 180-230 nm and 1 h after production is used for cell culture experiments immediately after production. The Complexes are in the cell culture medium M199 + used for the transfection 10% FCS stable (size 300-400 nm).

The complex formation can also take place by condensation of the DNA by PEI. For this purpose, both substances are first combined (one-time addition or in shares s. o.) and waited 15 min. After that, the liposomes pipetted in and the mixture pipetted up and down several times (preferably 10 ×). This complex is also left to stand for 15 minutes before use.  

Approach for a 3 cm cell culture dish

Endosomal delivery and transfection efficiency can be improved by a "fusion peptide", hemagglutinin (HA) derived from the membrane protein of the influenza virus [1, 2]. For this purpose, the peptide is added to the liposomes at the beginning of the complex formation and the mixture is then used like the pure liposomes according to the instructions. The concentration of the HA peptide can be between 0.1 and 1 nmol per batch.
[1] Wagner et al. (1992) Proc. Natl. Acad. Sci. USA 89 (17), 7934-7938
[2] Smoes and Slepushkin (1998) Gene Therapy 5 (7), 955-964

Example 2 Biological testing of the liposomes according to the invention 1) Investigations on culture cells

Human umbilical cord endothelial cells (HUVEC), melanoma cells (MeWo) and Fibroblasts (3T3) were grown in cell culture using the method known to those skilled in the art Cell culture technology increased and isolated.

Liposomal vector complexes with components a) + b) + c) + d) or (for control) with components a) + b) + c) (where component b) protamine sulfate instead PEI2000) were in excess (ratio about 20: 1) to the cells given and the mixture incubated at 37 ° C for 1 hour. Below were the Washed cells and incubated for another 60 hours in fresh cell culture medium. Subsequently, the successful uptake of the complexes into the cell, the Transcription and expression of the reporter gene in the plasmid by detection of the GFP autofluorescence.

The following results are achieved:
About 5% of the cells which were incubated with control liposomal vector complexes showed an expression of GFP. In HUVEC this expression rate was approximately the same as in melanoma cells and fibroblasts (3T3).

In contrast, only HUVEC showed that with the invention Liposomal vector complexes were incubated, a clear expression of GFP. More than 80% of the cells expressed GFP.

Claims (18)

1. Liposomal vector complex containing the following components

• a) a nucleic acid sequence of any length;
• b) a cationic carrier which condenses component a) and is at the same time lysosomotropic;
• c) lipids and phospholipids which form a liposome; and
• d) optionally a ligand which has a binding site for a target cell and is conjugated to a lipid; and
• e) optionally the functional sequence from the subunit HA-2 of the hemagglutinin of the influenza virus;

available through

• 1. Mixing component a) with component b) in the molar ratio [a): b)] from 1: 1 to 1: 1000, the mixing ratio being adjusted so that the net charge of the resulting overall complex is preferably either cationic or anionic, and
• 2. the complex resulting from step 1) is introduced into component c), and
• 3. optional component d) is inserted into component c); and
• 4. optional component e) is inserted into the complex resulting from step 2 or 3.

2. Liposomal vector complex according to claim 1, the isoelectric point in the pH range from 3-9.   3. Liposomal vector complex according to claim 2, the isoelectric point in the pH range is 3-7. 4. Liposomal vector complex according to one of claims 1-3, wherein the Component a) represents a DNA or RNA. 5. Liposomal vector complex according to one of claims 1-3, wherein the Component a) is a viral vector. 6. The liposomal vector complex according to claim 5, wherein the viral vector is selected from a group comprising RTV, AAV and Lenti V. 7. A liposomal vector complex according to any one of claims 1-6, in which the cationic carrier b) is a cationic polymer. 8. A liposomal vector complex according to claim 7, wherein the cationic Polymer is polyethyleneimine (PEI). 9. A liposomal vector complex according to claim 8, wherein the Molecular weight of the PEI is in the range of 500 to 20,000 Da. 10. A liposomal vector complex according to claim 9, wherein the PEI Molecular weight of 2000 Da on average. 11. The liposomal vector complex according to claim 10, wherein the target cell-specific Ligand is conjugated to a lipid. 12. Lipsomal vector complex according to any one of claims 1-11, wherein the target cell a tissue cell, an epithelial cell, an endothelial cell, a blood cell, one Is leukemia cell or a tumor cell.   13. A liposomal vector complex according to any one of claims 1-12, in which the isoelectric point of the complex is in the pH range of 4-7. 14. Liposomal vector complex according to one of claims 1-13 for the Transfection and transduction of a cell. 15. Use of a liposomal vector complex according to any one of claims 1-13 or a cell according to claim 14 for the manufacture of a remedy for the Prophylaxis or therapy for a disease. 16. Use of the liposomal vector complex according to claim 15 for Administration on the skin, on a mucous membrane, in a body cavity, in the Connective tissue, into an organ or into the bloodstream. 17. Liposomal vector complexes according to claims 1-16, in which in the Component c) a target cell-specific ligand is inserted. 18. A method for producing a liposomal vector complex according to any one of claims 1 to 14, wherein

• 1. Component a) according to claim 1 is mixed with component b) according to claim 1 in a molar ratio [a): b)] of 1: 1 to 1: 1000, the mixing ratio being adjusted so that the net charge of the resulting overall complex is preferred is either cationic or anionic;
• 2. the complex resulting from step 1 is introduced into component c) according to claim 1; and
• 3. optional component d) according to claim 1 is inserted into component c); and
• 4. optional component e) according to claim 1 is inserted into the complex resulting from step 2 or 3.