AU4758300A

AU4758300A - Novel liposomal vector complexes and their use in gene therapy

Info

Publication number: AU4758300A
Application number: AU47583/00A
Authority: AU
Inventors: Sabine Brusselbach; Alfred Fahr; Kristina Muller
Original assignee: Vectron Therapeutics AG
Current assignee: Vectron Therapeutics AG
Priority date: 1999-06-02
Filing date: 2000-05-23
Publication date: 2000-12-28
Also published as: EP1187929A2; WO2000074646A3; WO2000074646A2; DE19925143A1; JP2003501373A; CA2375854A1

Description

WO 00/74646 PCT/EP00/04678 Novel liposomal vector complexes and their use for gene therapy The essential components of vectors for gene therapy have hitherto been nucleic acid sequences which are complexed with a nonviral carrier (e.g. 5 cationic lipids or cationic polymers) or are inserted into a virus. Previous experience with such vectors in the gene therapy of all sorts of diseases, but in particular of oncoses, shows that, in cell culture, nonviral vector complexes particularly can only transduce a relatively low number of 10 cells (usually between 1% and 30%); furthermore that after administration of a gene therapy vector into the circulation of an organism these vectors are eliminated from the circulation in a short time and are no longer available for binding to the target cells and for the transfection of these target cells (Ogris et al. Gene Ther. 6: 595, 1999; Dash et al., Gene Ther. 15 6: 643, 1999; Li et al., Gene Ther. 5: 930, 1998; Liu et al. Gene Ther. 4: 517,1997). This elimination can take place due to degradation of the DNA or due to rapid deposition of the vectors in the lung, the liver or the 20 'reticuloendothelial system' (RES) which is particularly developed in the spleen and the lymph nodes (Zhu et al., Science 261: 209, 1993). The causes of the rapid elimination are varied. They can be: an excessively large negative or positive charge, an excessively large volume or an 25 opsonization of the vector particles by blood proteins. In the case of viral vectors, they can additionally be the binding of the virus coat proteins to virus-specific receptors in the organs and/or alternatively antibodies or immune cells specific for the viruses which bind to the vectors and thereby eliminate these. 30 Previous experience additionally shows that the coupling or insertion of a target cell-specific ligand into the vector complex does not significantly decrease its rapid elimination after administration into the blood circulation. 35 In the knowledge of these problems, the urgent need exists for novel preparations of vectors which transfects as many cells as possible in the cell culture and which, after administration to a living organism, remain as long as possible in the circulation and are not prematurely eliminated from the circulation. In order to decrease the elimination of cationic lipids or 2 cationic polymers as a complex with nucleic acid sequences from the blood circulation, 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 (Torchilin et al., Biochim. Biophys. Res. 5 Acta 1195: 181, 1994) or other amphipathic polymers (Woodle et al., Bioconjugat. Chem. 5: 493, 1994) were coupled to the cationic lipids or cationic polymers or, with the aid of negatively charged lipids, anionic liposomes were prepared in which the nucleic acid sequences were included as a complex with cationic lipids or cationic polymers (US Patent 10 No. 4,946,787; US Patent No. 4,245,737; US 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: 15 485,1998). Modifications of this type led, for example, to a stabilization of the vector particle size, inhibited the aggregation of vectors with themselves or with blood cells, reduced the opsonization of vectors by binding of 20 immunoglobulins, complement fractions, fibrinogen or fibronectin, protected (adeno)viral vectors against elimination by antibodies (Chillon et al., Gene Ther. 5: 995, 1998) and caused a prolongation of the blood residence time of vectors, a markedly stronger concentration in tumors growing subcutaneously and a transduction of the tumor cells (Ogris et al., Gene 25 Ther. 6: 595, 1999). At the same time, however, it was also possible in the lung, spleen and liver to detect a considerable concentration of the vectors and transduction of the tissue cells in these organs (Ogris et al., Gene Ther. 6: 595, 1999), 30 so that it can be concluded that, for example, the coupling of PEG does bring about an improvement, but still no optimization of the distribution of vectors. General description of the invention 35 The invention relates to novel liposomal vector complexes for gene therapy consisting of the following components: a) a nucleic acid sequence of any desired length; 3 b) a cationic carrier which condenses component a) and is lysosomolytic and/or lysosomotropic; c) lipids and phospholipids which form a liposome; d) optionally a ligand which has a binding site for a target cell; 5 e) optionally a fusogenic substance which can replace the lysosomolytic and/or lysosomotropic function of component b); where in the presence of a fusogenic substance(s) the cationic carrier (b) must not be lysosomolytic and/or lysosomotropic. 10 Component a) can be a nonmodified or modified DNA sequence or a nonmodified or modified RNA sequence. The nucleotide sequence can exert an anti-DNA (triplex) or anti-RNA (antisense; ribozyme) function or can code for an active RNA sequence of this type or for a protein. The nucleotide sequences and their modification can be such that the 15 nucleotide sequence is largely resistant to degradation by DNAses or RNAses. Examples of nucleotide sequences of this type 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, 20 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 additionally be a virus, preferably a virus in which a 25 nucleic acid sequence foreign to the virus has been inserted using the methods known to the person skilled in the art. Examples of viruses of this type are RTV, AAV and lentiviruses. Examples of this type and further examples have been described by Vile, Nature Biotechnol. 15: 840, 1997; McKeon et al., Human Gene Ther. 7: 1615, 1996; Flotte et al., Gene Ther. 30 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 condenses component a) and at the same time has lysosomolytically and/or lysosomotropically and/or 35 lysosomotropic properties. According to this invention, in a particular embodiment component b) is a cationic polymer, for example described by Boussif et al., Proc. Nati. Acad. Sci. USA 92: 7297, 1995; Kaneda et al., Science 243: 375, 1989; Keown et 4 al., Methods in Encymology 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. The polyethyleneimine (PEI) described by Boussif, when used as a 5 vector for gene therapy according to the method described by the same author, finally leads to a swelling and bursting of the lysosomes, i.e. PEI acts lysosomolytically. In a further particular embodiment of this invention, component b) is a 10 polyethyleneimine (PEI), in a further particular embodiment of this invention the polyethyleneimine has a molecular weight in a range of 500-25,000 Da and in a further embodiment a molecular weight of 5000-10,000 Da in a further embodiment of the invention a molecular weight of on average approximately 2000 Da and was prepared as described in the patent 15 application EP-A 0 905 254. High-branched-chain PEI (Lupasol@, BASF, Ludwigshafen, Germany) and a low-branched-chain PEI derivative, which was prepared according to Fischer et al. (Pharm. Res. 16, 1273-1279, 1999), are used in a particular embodiment of the invention. 20 Component c) is any desired liposome having any desired composition known to the person skilled in the art. In a particular embodiment, this liposome has an anionic charge. In a further particular embodiment the lipid and phospholipid composition of the anionic liposome is similar to the composition of a virus coat. The preparation of liposomes having anionic 25 charge has already been widely described, for example from in US patents Nos. US 4,946,787, US 4,245,737, US 5,480,463, and also in Heywood and Eanes, CaIc. 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 30 al., J. Lip. Res. 8: 114, 1998; Schoen et al., J. Lip. Res. 8: 485, 1998. The preparation of liposomes which are similar to virus coats was described, for example, in the US patents Nos. US 5,252,348; US 5,753,258; US 5,766,625 and EP-A 0 555 333. 35 The invention additionally relates to the completion of the liposomal vector complexes according to the invention by addition of a component d).

5 This component d) is a ligand which has a binding site for the target cell and is conjugated to a lipid. The target cell specificity of the ligand can be arbitrary. 5 Preferred target cell specificities of ligands are selected from a group described in detail in - polyfunctional ligand systems for the target cell-specific transfer of nucleotide sequences EP-A 0 846 772 10 - single-chain, double antigen-binding molecules (DE 198161417, still unpublished) - specific cell membrane-penetrating molecules (DE 19850987.1, still unpublished) or - target cell-specific, multivalent proteins (DE 19910419.0, still 15 unpublished). The type of lipid can be arbitrary, but naturally occurring lipids, such as described, for example, in US patent Nos. US 5,252,348; US 5,753,258; US 5,766,625 and EP-A 0 555 333 are preferred. 20 The conjugation of lipids to the target cell-specific ligand is carried out using one of the methods known to the person skilled in the art, for example as described in US patent No. US 5,662,930. 25 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 US patents Nos. 5,252,348 and US 5,753,258). 30 The invention additionally relates to the completion of the liposomal vector complexes according to the invention by addition of a component e). This component e) is the functional sequence of a fusion peptide, preferably from subunit HA-2 of the hemagglutinin from the influenza virus (Wagner et al., Proc. NatI. Acad. Sci. USA 89(17), 7934-7938) ligands, which facilitate 35 the release of the liposome contents from the endosome. The preparation of the vector complex according to the invention consisting of the components a), b) and c) or a), b, c) and d) or a), b), c), d) and e) is 6 carried out using methods known to the person skilled in the art, for example in such a way that - in the 1 st step component a) is mixed with component b), the mixing 5 ratio being adjusted such that the net charge of the resulting total complex is preferably either cationic or anionic and subsequently - in the 2 nd step the complex resulting from step (1) is inserted into the component c), which can already contain component d), the mixing 10 ratio of all components being adjusted such that the net charge of the resulting overall complex is preferably either anionic or cationic; - in a 3 rd step component d) can also optionally be inserted into component c) following step 2; and 15 - in a 4 th step component e) is optionally inserted into the complex resulting from step 2 or 3, or, alternatively, component e) is added to component c) before step 2. 20 The liposomal vector complexes resulting from these preparation steps have a diameter of 100 - 600 nm and a cationic or anionic charge, preferably a diameter of 100 - 300 nm and an anionic charge. The liposomes according to the invention are enriched, for example, in the 25 tumor vascular bed (Unezaki et al., Int. J. Pharmac. 114:11, 1996; Sadzuka et al., Cancer Lett. 127:99, 1998; Wunder et al., Int. J. Oncol. 11:497, 1997). In addition, the liposomes according to the invention bind via their component d) to the target cell and transfects this such that the nucleic acid sequence in the vector complex according to the invention is released in 30 the cell. This nucleic acid sequence can display its action, depending on composition, in the target cell, i.e. for example inhibits the transcription or translation of a certain gene or of a certain RNA, or transduce the cell for 35 the expression of the RNA or of the protein encoded by this nucleic acid sequence. The transduction rate by the liposomal vector complexes according to the invention is considerably improved in comparison to the existing technique 7 known to the person skilled in the art and is, for example in the cell culture, over 80% of the cells which carry a receptor for component d) and are brought into contact with the liposomal vector complexes according to the invention. 5 The vector complexes according to the invention are thus preferably suitable for in vitro transduction of cells and for in vivo administration with the aim of prophylaxis or therapy of diseases. 10 The invention relates to a liposomal vector complex comprising the following components: f) a nucleic acid sequence of any desired length; g) a cationic carrier which condenses component a) and is lyosomolytic and/or lysosomotropic; 15 h) lipids and phospholipids which form a liposome; i) optionally a ligand which has a binding site for a target cell; j) optionally a fusogenic substance which can replace the lysosomolytic and/or lysosomotropic function of component b); where in the presence of a fusogenic substance(s) the cationic carrier (b) 20 must not be lysosomolytic and/or lysosomotropic; and in which component a) is preferably a polynucleic acid, component b) a cationic protein, a cationic polymer or a combination of both. In a further embodiment of the invention, the cationic carrier is protamine 25 sulfate. In a further embodiment of the invention, component b) is a cationic polymer, in particular polyethyleneimine (PEI), particularly preferably PEI having a molecular weight of on average 2,000 - 10,000 Da, very 30 particularly preferably a high-branched-chain or low-branched-chain PEI. In a further embodiment of the invention, component c) is constructed of phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, anchor lipid, and cholesterol, a particularly preferred anchor lipid is an N 35 carboxyphosphatidylethanolamine, e.g. an N-glutarylphosphatidylethanol amine; and component d) is preferably conjugated to one of the components a) - c) without an anchor, via an anchor or via an anchor lipid.

8 A further embodiment of the invention is a liposomal vector complex in which component d) is embedded noncovalently in the liposome surface. A further embodiment of the invention is a liposomal vector complex whose 5 target cell is a tissue cell, an epithelial cell, an endothelial cell, a blood cell, a leukemia cell or a tumor cell. A further embodiment of the invention is a liposomal vector complex whose component e) is the functional sequence from the subunit HA-2 of the 10 hemagglutin of the influenza virus or a synthetic derivative thereof. A further embodiment of the invention is a liposomal vector complex for the transduction and transfection of cells in vitro or in vivo, serum preferably being used in vitro. 15 A further embodiment of the invention is the use of a liposomal vector complex for the production of a diagnostic for use in vitro and in vivo and/or for the production of a therapeutic for the prophylaxis or therapy of a disease in vivo and ex vivo, administration preferably taking place on the 20 skin, on a mucus membrane, in the lung, on the eye, in a body cavity, in the connective tissue, in the muscle, in an organ or in the blood circulation. A further embodiment of the invention is a process for the preparation of a liposomal vector complex, where 25 (1) component a) is mixed with component b), (2) the complex resulting from step (1) is introduced into component c), the mixing ratio of all components being adjusted such that the net charge of the resulting overall complex is preferably 30 either cationic or anionic; (3) component d) is optionally inserted into component c) before or after complex formation; (4) component e) is optionally inserted into the complex resulting from steps (2) and (3) or into component c) before complex 35 formation; the resulting product preferably being lyophilized or aerosolized. Examples for illustration of the concept of the invention 9 The following examples are intended to illustrate how the present invention could be carried out. Example 1: 5 Modified peptide having an RGD sequence For the improvement of the targeting of the integrin receptors, a cyclic peptide was synthesized. It is a CDCRGDCFC peptide (Arap W., Pasqualini, R. and Ruoslahti, E. (1998) Science, 279: 377-380; Pasqualini, 10 R. Koivunen, E. Ruoslathi E., Nature Biotech. (1997) 15:542-546) having an additional arginine at the N-terminal end (FW 1163.35). The terminal amino acid reduces the extent of the coupling of the peptide to the active center, the RGD sequence. 15 The cyclization takes place by means of oxidation of the thiol group to disulfide bridges. Successful cyclization is checked by means of HPLC analysis. After the HPLC purification, the peptide is lyophilized, stored at 4*C and dissolved in buffer (250 tg/150 pl of tris buffer 10 mM pH 7.4 or PBS buffer 20 pH 7.4) before use. Preparation of the liposomes Material 25 Substance Manufacturer Batch Parts FW DOPS Avanti 181PS-P36a 3 810 Sodium salt DLPE Sygena 0998 3 579.76 Cholesterol Calbiochem 228111 3 386.7 N-glut-PE in-house synthesis 16118 1 805.97 or Avanti 30 10 Stock solutions Substance Concentration Batch Vol. Amount of [Eimol/ml] employed substance [Il] employed [pmol] DOPS 16.13 15128 186 3 DLPE 33.47 09039 89.7 3 Cholesterol 24.95 09039 120.2 3 N-glt-PE 2 16118 500 1 The liposomes are prepared by the film/hydration method. The lipids 5 dissolved in chloroform and the lipid anchors are pipetted into a 100 ml round-bottomed flask and the chloroform is stripped off for 15 min. The flask dips during the course of this into a temperature-controlled water bath having a temperature which is above the phase-transition temperature of the lipids, in this specific case 30 0 C. For the complete removal of the 10 solvent, the film is dried in a high vacuum for 15 min. Hydration of the film with buffer follows (tris 10 mM, pH 7.4 or PBS pH 7.4, other buffers and pHs are likewise possible). The buffer is added to the flask together with a few small glass beads and the batch is rotated for 45 15 min with N 2 aeration, the flask also dips into the warm water bath at 30 0 C here. For the swelling of the lipid film and for the production of multilamellar liposomes (MLV), the batch is allowed to stand at room temperature for 2 hours [1, p.38]. The MLV suspension is transferred to a special sonicator glass vessel and the batch is sonicated for 15 sec by 20 means of a probe sonicator (here Soniprep 150 (preferably adjusted amplitude in microns 8-12)). The suspension dips into an ice bath in the course of this. After the sonication, a pause of 30 sec is inserted for the cooling of the suspension. This procedure (sonication - pause) is repeated 10 times. The size measurement of the SUV obtained affords a size of 120 25 to 300 nm. After subsequent extrusion [1, pp.52-56][2] by means of 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 prepared are stable for at least 2 months and do not measureably change their size in this time. The bottling of the liposomes for further processing is 30 carried out under an air hood in a sterile Eppendorf cap.

11 References [1] Roger R.C. New, "Preparation of liposomes", chapter 2, Liposomes a practical approach (1989) [2] Olson et al. (1980) Biochim. Biophys. Acta, 394, 483 5 Coupling of the peptide to lipid anchors (Weissig, V., Lasch, J., Klibanov, A.L. Torchilin, V.P., A new hydrophobic anchor for the attachment of proteins to liposomal membranes. FEBS Lett. 202, 1986, 86-90; Bogdanov et al., "Protein immobilization on the surface of liposomes via carbodiimide 10 activation in the presence of N-hydroxysulfosuccinimide", FEBS Lett. 231, 1988, 381-384; Weissig, Qualifying thesis for university lecturers "Methoden zur Darstellung funktionalisierter Liposomen mit Adjuvanseffekt" [Methods for the preparation of functionalized liposomes having an adjuvant effect] MLU Halle-Wittenberg (1992); Thesis Ragna Schmidt, 15 Halle University (1997). [3] Martin et al. "Covalent attachment of protein to liposomes", chapter 4, Roger R.C. New, Liposomes a practical approach (1989)) Material 20 Substance Conc. Batch Solvent Amount Amount of mark employed substance employed Liposomes 10 ptmol/ml 12039 tris buffer 400 I1 4 ltmol pH 7.4 EDAC pure 25H0993, solid 3.5 mg 18.26 substance Sigma p[mol RGD 250 gg/ 22049 tris buffer 150 W= 0.215 solution 150 d pH 7.4 250 pg Rmol Firstly, the carboxyl group of the glutaric acid radical of the lipid anchor is activated by the addition of 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide [3, p. 170]. For this, 400 pl of the liposome suspension (4 ptmol of total lipid, 25 0.4 ptmol of N-glut-PE, medium tris buffer pH 8, 10 mM, other buffers and pHs are likewise possible) are vortexed and 3.5 mg of EDC are weighed in. The batch is again vortexed and shaken for 5 hours protected against light (IKA Vibrax VXR). The activated intermediate, the O-acyl intermediate, is formed, to which the peptide having a free amino function binds with 30 formation of an amide. 250 [tg of RGD dissolved in PBS buffer pH 7.4 or 12 tris buffer 10 mM pH 7.4 (250 pg/l50 pi, other buffers and pHs are likewise possible) are added, and the batch is vortexed and shaken overnight. After coupling has taken place, the unbound peptide is separated from the liposomes by gel chromatography. Size exclusion chromatography is 5 carried out using a Sephadex G 50 column, the eluent is tris buffer 10 mM, pH 7.4 (other buffers and pHs are likewise possible). The coupling yield is carried out by means of a simultaneously carried out batch containing fluorescent dye (5-DTAF)-labeled RGD (Product 10 information sheet 5-DTAF, Molecular Probes (MP 00143 08/27/95) "Conjugation with Amine-Reactive Probes") and is at least 6 [tg of RGD/ 1 pmol of PL (calculated for actual amounts of lipid using cholesterol). The determination of the coupling efficiency can also alternatively be carried out by means of HPLC (Gyongyossy-Issa et al. "The Covalent Coupling of Arg 15 Gly-Asp-Containing Peptides to Liposomes: Purification and Biochemical Function of the Lipopeptide" Archives of Biochemistry and Biophysics, Vol. 353, No. 1, May 1 (1998)). The size of the liposomes coupled using RGD is between 100-150 nm. The coupling can be modified according to Bogdanov in order to increase the coupling efficiency. The coupling can 20 also be carried out on its own using an anchor lipid according to the process described by Weissig. The resulting lipid anchor-peptide construct can be employed, like the lipid, in the preparation of liposomes. Example 2: Preparation of a liposomal vector complex according to the 25 invention in the sequence plasmid, liposome, protamine sulfate and PEI The plasmid was condensed with PEI, which had been prepared according to the method described by Fischer et al. (Pharm. Res. 16, 1273-1279, 1999), or using Lupasol (BASF, Ludwigshafen, Germany). 30 Complex formation is firstly carried out by mixing together the negatively charged constituents plasmid DNA (pG13, Clontech, Heidelberg, Germany) and liposomes (DLPE, DOPS, cholesterol, N-glutaryl-PE 3:3:3:1). In this process, the dilution of the solutions is to be taken into account in order to 35 prevent irreversible precipitate formation. The final volume of all constituents mentioned is 100 pl. Firstly, the buffer (tris 10 mM, pH 7.4, other buffers and pHs are likewise possible) is introduced and 10 pg of plasmid (10 stg/60 d) and also 40 Ig of liposomes (variable, between 1 and 6 ptg/i) are mixed together by simple pipetting. The mixture is vortexed.

13 The condensation of the DNA is then carried out by addition of the cationic agent, firstly 19.96 stg of protamine sulfate is added (charge ratio +/- 3.3:1). For this, the protamine sulfate is added rapidly thereto using an Eppendorf pipette and the batch is mixed by pipetting to and fro 10 times and a 5 coating of the complex with the lipids is achieved. 29.7 gg (amounts up to 16.2 ltg are likewise possible) of PEI (N/P ratio 20.7, reduction to 7.5 possible), a further cationic agent, are then added. For this, 33 I of PEI solution 0.9 mg/ml are diluted with 250 pl of high-purity water and added to the batch in substeps. The addition is carried out in 2 x 100 pl and 1 x 85 Iil 10 steps, pipetting the batch to and fro 10 times each after the addition and waiting for 15 min. A complex of this type has a size of 180-300 nm 1 h after the preparation and is used immediately after the preparation for the cell culture experiments. The complexes are stable in the cell culture medium M199 + 10% FCS used for the transfection (size 360-500 nm). 15 Example 3: Preparation of a liposomal vector complex according to the invention in the sequence plasmid, liposome, and PEI The batch as described in Example 2 can also be prepared without 20 protamine sulfate. The preparation steps are identical, with the exception of the addition of 19.96 lag of protamine sulfate. Transfection experiments show almost identical efficiency. Example 4: Preparation of a liposomal vector complex according to the 25 invention in the sequence plasmid, liposome, fusion peptide, and PEI The preparation is carried out according to the method and sequence as described in Example 3. In addition, a "fusion peptide", hemagglutinin (HA) originating from the membrane protein of the influenza virus (Wagner et al., 30 Proc. Natl. Acad. Sci. USA 89, 7934-7938, 1992; Smoes & Slepushkin Gene Therapy 5, 955-964, 1998), is added to the liposomes at the start of complex formation and the mixture is then used like the pure liposomes according to Example 3. The concentration of the HA peptide can be between 0.1 and 1 nmol (1 - 10 pig) per batch. 35 Example 5: Preparation of a liposomal vector complex according to the invention in the sequence plasmid, PEI, liposome, fusion peptide.

14 Complex formation can likewise be carried out by condensation of the DNA by PEI. For this, firstly both substances are mixed together (single addition or in portions, see above) wait for 15 min. The liposomes are then pipetted in and the mixture is pipetted to and fro several times (preferably 10 x). 5 This complex is likewise allowed to stand for 15 min before use. The amount of liposomal formulation of plasmid, cationic agent and liposomes needed for the transfection can be reduced to 5 ltg of plasmid/3 cm dish. In addition, the volume of the formulation can be decreased. 10 Complex formation is firstly carried out by mixing together the constituents plasmid DNA and PEI. In this process, the dilution of the solutions is to be taken into account in order to prevent irreversible precipitate formation. The final volume of the two constituents mentioned is 245.93 I. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs are likewise possible) is 15 introduced and 15 ptg of plasmid (15 ptg/90 pal) are added thereto. The condensation of the DNA is then carried out by addition of the cationic agent, of 44.55 jig of PEI (N/P ratio 20.7, larger amounts and a reduction to 9.79 jg are likewise possible). For this, 49.5 Il of PEI solution 0.9 mg/mI are diluted with 106.43 pl of high-purity water and added to the batch in 20 substeps. The addition is carried out in 1 x 100 Il and 1 x 55.93 pil steps, the batch is pipetted to and fro 5 x after the addition and finally pipetted to and fro 5 x with a 100 pl volume, wait for 15 min. 60 jig of liposomes (variable, between 1 and 6 ptg/pl) are mixed with 15 pig of HA fusion peptide (reduction of the amount to 0.1 jg possible) and added to the plasmid/PEI 25 complex by simple pipetting. For this, the batch is pipetted to and fro 10 x with a 100 Il volume after the addition. The mixture is vortexed. Complexes of similar activity are also obtained by simple mixing of all components indicated. After preparation, a complex of this type has a size of 180-250 nm and is used immediately after preparation for the cell culture 30 experiments. The complexes are stable in the cell culture medium M199 + 10% FCS used for the transfection (size 300-400 nm). Example 6: Preparation of a liposomal vector complex according to the invention in the sequence plasmid, PEI, liposome 35 The liposomal formulation can be prepared without the HA fusion peptide. For this, the abovementioned preparation procedure is used and only the addition of the HA peptide is omitted.

15 Example 7: Preparation of a liposomal vector complex according to the invention in the sequence plasmid, PEI, liposome, with and without HA peptide, with variable volumes. 5 Complex formation is firstly carried out by mixing together the constituents plasmid DNA and PEI. In this process, the dilution of the solutions is to be taken into account in order to prevent irreversible precipitate formation. The final volume of the two constituents mentioned is, depending on the batch type: ~ a) 465 p.1, b) 245.93 R1, c) 196.4 pI, d) 150 p1. Firstly, the buffer (tris 10 10 mM, pH 7.8, other buffers and pHs are likewise possible) is introduced and 15 pg of plasmid (15 pig/90 p1 or 75 [d) added thereto. The condensation of the DNA is then carried out by addition of the cationic agent, of 44.55 pg of PEI (N/P ratio 20.7, larger amounts and a reduction to 9.79 lag are likewise possible). For this, 49.5 p of PEI solution 0.9 mg/mI 15 are diluted with high-purity water (325.5, 106.43, 56.9, 25.5 p) and added to the batch in substeps. The addition is carried out in 100 pl and/or variable (55.9, 75, 106 pl) steps, after the addition the batch is pipetted to and fro 5 x each and finally pipetted to and fro 5 x with a 100 p volume, wait for 15 min. 60 gg of liposomes (variable, between 1 and 6 Rg/pl) are 20 mixed with 15 jig of HA fusion peptide (reduction of the amount to 0.1 ptg possible). This addition of the HA fusion peptide is optional, it can be omitted without a relatively large decrease in transfection. The liposomes are added to the plasmid/PEI complex by simple pipetting. For this, the batch is pipetted to and fro 10 x after the addition with a 100 pl volume. 25 The mixture is vortexed. Complexes of similar activity are also obtained by simple mixing of all components indicated. After preparation, a complex of this type has a size of 180-250 nm and is used immediately after preparation for the cell culture experiments. The complexes are stable in the cell culture medium M199 + 10% FCS used for the transfection (size 30 300-400 nm). Example 8: Preparation of a liposomal vector complex according to the invention in the sequence plasmid, protamine sulfate, PEI and liposome. 35 Complex formation is firstly carried out by mixing together the constituents plasmid DNA and protamine sulfate. In this process, the dilution of the solutions is to be taken into account in order to prevent irreversible precipitate formation. The final volume of the two constituents mentioned is, depending on the batch type: ~ a) 369 p.1, b) 320 pl. Firstly, the buffer (tris 16 10 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and 15 pg of plasmid (15 ptg/90 l) added thereto. The condensation of the DNA is then carried out by addition of the protamine sulfate (+/- ratio 3.3, larger or smaller amounts are likewise possible). Protamine sulfate is diluted with 5 high-purity water (29.94 ptg/105 Itl) and added to the DNA, the batch being pipetted to and fro 10 x. The complex is allowed to stand for 15 min for maturation. 49.5 tl of PEI solution 0.9 mg/ml are then diluted with high purity water (106.43, 56.9 pl) and added to the batch in substeps. The addition is carried out in 100 pl and/or 55.9 pl steps, after the addition the 10 batch is pipetted to and fro 5 x each and finally pipetted to and fro 5 x with a 100 pl volume, wait for 15 min. 60 gg of liposomes (variable, between 1 and 6 gg/pl) are mixed with 15 mg of HA fusion peptide (reduction of the amount to 0.1 pg possible). This addition of the HA fusion peptides is optional, it can be omitted without a relatively large increase in transfection. 15 The liposomes are added to the plasmid/PS/PEI complex by simple pipetting. For this, the batch is pipetted to and fro 10 x after the addition with a 100 p volume. The mixture is vortexed. Complexes of similar activity are also obtained by simple mixing of all components indicated. After preparation, a complex of this type has a size of 180-300 nm and is used 20 immediately after preparation for the cell culture experiments. The complexes are stable in the cell culture medium M199 + 10% FCS used for the transfection (size 300-500 nm). These complexes can be prepared with other charge ratios, it being possible to vary both the proportion of the PS and of the PEI. In this case, larger or smaller volumes can likewise be 25 chosen. Example 9: Preparation of a liposomal vector complex according to the invention in the sequence plasmid, protamine sulfate, PEI and liposome having a reduced PEI content. 30 Complex formation is firstly carried out by mixing together the constituents plasmid DNA and protamine sulfate. In this process, the dilution of the solutions is to be taken into account in order to prevent irreversible precipitate formation. The final volume of the two constituents mentioned is, 35 depending on the batch type: ~ 319 pl. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and 15 Rg of plasmid (15 pg/90 pl) are added. The condensation of the DNA is then carried out by addition of the protamine sulfate (+/- ratio 3.3, larger or smaller amounts are likewise possible). Protamine sulfate is diluted with 17 high-purity water (29.94 ptg/l05 pl) and added to the DNA, the batch being pipetted to and fro 10 x. The complex is allowed to stand for 15 min for maturation. 30 l (N/P ratio 12.5) or 18 pil (N/P ratio 7.5) of PEI solution 0.9 mg/ml are then diluted to 106 pl with high-purity water (76 or 88 pl) and 5 added to the batch in substeps. The addition is carried out in 106 pl steps, after the addition pipette the batch to and fro 10 x each, wait for 15 min. 60 jig of liposomes (variable, between 1 and 6 pg/pl) are mixed with 15 jg of HA fusion peptide (reduction of the amount to 0.1 pg possible). This addition of the HA fusion peptides is optional, it can be omitted without a 10 relatively large decrease in transfection. The liposomes are added to the plasmid/PS/PEI complex by simple pipetting. For this, the batch is pipetted to and fro 10 x with a 100 Il volume after addition. The mixture is vortexed. Complexes of similar activity are also obtained by simple mixing of all components indicated. 15 Example 10: Preparation of a liposomal vector complex according to the invention in the sequence plasmid, PEI, protamine sulfate and liposome. Complex formation is firstly carried out by mixing together the constituents 20 plasmid DNA and PEI. In this process, the dilution of the solutions is to be taken into account in order to prevent irreversible precipitate formation. The final volume of the two constituents mentioned is, depending on the batch type: ~ 246 pl. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and 15 jig of plasmid (15 pg/90 pal) are 25 added thereto. The condensation of the DNA is then carried out by addition of 44.55 jig of PEI (N/P ratio 20.7, larger amounts and a reduction are likewise possible). For this, 49.5 pl of PEI solution 0.9 mg/ml are diluted with high-purity water (to 155.93 pl) and added to the batch in substeps. The addition is carried out in 100 ji and 55.9 jil steps, after the addition the 30 batch is pipetted to and fro 5 x each and finally pipetted to and fro 5 x with a 100 sl volume, wait for 15 min. The protamine sulfate (+/- ratio 3.3, larger or smaller amounts are likewise possible) is then added. Protamine sulfate is diluted with high-purity water (29.94 jig/105 jil) and added to the DNA, the batch being pipetted to and fro 10 x. The complex is allowed to stand 35 for 15 min for maturation. 60 jig of liposomes (variable, between 1 and 6 jig/il) are mixed with 15 jig of HA fusion peptide (reduction of the amount to 0.1 jig possible). This addition of the HA fusion peptides is optional, it can be omitted without a relatively large decrease in transfection. The liposomes are added to the plasmid/PEI/PS complex by simple pipetting.

18 For this, the batch is pipetted to and fro 10 x after the addition with a 100 RI volume. The mixture is vortexed. Complexes of similar activity are also obtained by simple mixing of all components indicated. 5 Example 11: Preparation of a liposomal vector complex according to the invention in the sequence plasmid, PEI, protamine sulfate and liposome having a reduced PEI content. Complex formation is firstly carried out by mixing together the constituents 10 plasmid DNA and PEI. In this process, the dilution of the solutions is to be taken into account in order to prevent irreversible precipitate formation. The final volume of the two constituents mentioned is, depending on the batch type: ~ 196 I. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and 15 pg of plasmid (15 pg/90 [tl) are 15 added. The condensation of the DNA is then carried out by addition of 9.75 pg, 16.2 ptg or 27 ptg of PEI (N/P ratio 4.5, 7.5, 12.5, larger amounts and a reduction are likewise possible, e.g. N/P ratio 1.8). For this , 10.86 pl 18 I or 30 pl of PEI solution 0.9 mg/ml are diluted with high-purity water (to 106 pil) and added to the batch while pipetting to and fro 10 x. The 20 protamine sulfate is then added (+/- ratio 3.3, larger or smaller amounts are likewise possible). Protamine sulfate is diluted with high-purity water (29.94 jig/l05 il) and added to the DNA/PEI complex, the batch being pipetted to and fro 10 x. The complex is allowed to stand for 15 min for maturation. 60 jig of liposomes (variable, between 1 and 6 pig/pil) are mixed 25 with 15 jig of HA fusion peptide (reduction of the amount to 0.1 jpg possible). This addition of the HA fusion peptides is optional, it can be omitted without a relatively large decrease in transfection. The liposomes are added to the plasmid/PEI/PS complex by simple pipetting. For this, after the addition the batch is pipetted to and fro 10 x with a 100 ji volume. 30 The mixture is vortexed. Complexes of similar activity are also obtained by simple mixing of all components indicated. Example 12: Preparation of a liposomal vector complex according to the invention in the sequence plasmid, PEI and liposome having an increased 35 and reduced lipid content Complex formation is firstly carried out by mixing together the constituents plasmid DNA and PEI. In this process, the dilution of the solutions is to be taken into account in order to prevent irreversible precipitate formation. The 19 final volume of the two constituents mentioned is 245.93 pl. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and 15 [tg of plasmid (15 tg/90 jil) are added thereto. The condensation of the DNA is then carried out by addition of the cationic 5 agent, 44.55 ig of PEI (N/P ratio 20.7, larger amounts and a reduction are likewise possible). For this, 49.5 [I of PEI solution 0.9 mg/ml are diluted with 106.43 lI of high-purity water and added to the batch in substeps. The addition is carried out in 1 x 100 Vil and 1 x 55.93 d steps, after the addition the batch is pipetted to and fro 5 x each and finally pipetted to and fro 5 x 10 with a 100 [I volume, wait for 15 min. 75, 45 and 30 jig of liposomes (between 1 and 6 pg/pl) are added to the plasmid/PEI complex by simple pipetting. For this, the batch is pipetted to and fro 10 x after the addition with a 100 pal volume. The content of the liposomes used can moreover be markedly increased to 10 x the amount. 15 Example 13: Biological testing of the liposomal vector complexes according to the invention in various cell cultures Complex formation is firstly carried out by mixing together the constituents 20 plasmid DNA and PEI. In this process, the dilution of the solutions is to be taken into account in order to prevent irreversible precipitate formation. The final volume of the two constituents mentioned is 245.93 pl. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and 15 Ig of plasmid (15 ptg/90 Ipl) are added thereto. The 25 condensation of the DNA is then carried out by addition of the cationic agent, 44.55 Ig PEI (N/P ratio 20.7, larger amounts and a reduction are likewise possible). For this, 49.5 Il of PEI solution 0.9 mg/ml are diluted with 106.43 Il of high-purity water and added to the batch in substeps. The addition is carried out in 1 x 100 d and 1 x 55.93 ptl steps, after the addition 30 the batch is pipetted to and fro 5 x and finally pipetted to and fro 5 x with a 100 il volume, wait for 15 min. 60 jig of liposomes (between 1 and 6 Ig/pl) with and without coupled RGD targeter (RGD binds to the aN receptor) are added to the plasmid/PEI complex by simple pipetting. For this, the batch is pipetted to and fro 10 x after the addition with a 100 Il volume. 35 The batch is divided into 3 aliquots and one aliquot each is added to a 3 cm dish. The triplicate batch was added to a 10 cm dish for FACS analysis. The liposomal vector complexes, prepared as in Examples 1-11, were added to the cells (in 10 cm dishes for FACS analysis; 3 cm dishes for luciferase and GFP microscopy) and incubated at 37 0 C for 1-6 hours.

20 Subsequently, the cells were washed and incubated for a further 24-48 hours in fresh cell culture medium. The successful absorption of the complexes into the cells, the transcription, and the expression of the reporter gene in the plasmid by the detection of the GFP autofluorescence, 5 luciferase assay, and FACS analysis were then measured. Results of the FACS analysis are compiled in the following table. Cells + RGD -RGD HUVEC primary endothelial cells 73 29 MeWo melanoma cells 66 5 MSM melanoma cells 37 12 HMB-2 melanoma cells 30 7 B254 melanoma cells 10 4 DX-3 melanoma cells 11 3 Saos-2 osteosarcoma cells 15 28 DU-145 prostate carcinoma cells 1 1 PC3 prostate carcinoma cells 95 21 HeLa cervical carcinoma cells 4 33 LoVo colon carcinoma cells 8 1 A549 lung carcinoma cells 23 11 MCF-7 breast cancer cells 83 34 JEG-3 chorionic carcinoma cells 4 9

Claims

1. A liposomal vector complex comprising the following components a) a nucleic acid sequence of any desired length; 5 b) a cationic carrier which condenses component a) and is lysosomolytic and/or lysosomotropic; c) lipids and phospholipids which form a liposome; d) optionally a ligand which has a binding site for a target cell; e) optionally a fusogenic substance which can replace the 10 lysosomolytic and/or lisosomotropic function of component b); where in the presence of a fusogenic substance (e) the cationic carrier (b) must not be lysosomolytic and/or lysosomotropic.

2. A liposomal vector complex as claimed in claim 1, in which 15 component a) is a polynucleic acid.

3. A liposomal vector complex as claimed in claim 1 or 2, in which component b) is a cationic protein. 20

4. A liposomal vector complex as claimed in claim 3, in which the cationic protein is not lysosomolytic and is selected from a group comprising protamine sulfate.

5. A liposomal vector complex as claimed in claim 1, in which 25 component b) is a cationic polymer.

6. A liposomal vector complex as claimed in claim 5, in which the cationic polymer is polyethyleneimine (PEI). 30

7. A liposomal vector complex as claimed in claim 6, in which the PEI has a molecular weight of on average 2,000 - 10,000 Da.

8. A liposomal vector complex as claimed in claim 6, in which the PEI is high-branched-chain. 35

9. A liposomal vector complex as claimed in claim 6, in which the PEI is low-branched-chain. 22

10. A liposomal vector complex as claimed in claim 4 in which a PEI is additionally present.

11. A liposomal vector complex as claimed in claim 1, in which 5 component c) consists of phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, anchor lipid and cholesterol.

12. A liposomal vector complex as claimed in claim 11, the anchor lipid 10 being an N-carboxylphosphatidylethanolamine.

13. A liposomal vector complex as claimed in claim 12, the anchor lipid being an N-glutarylphosphatidylethanolamine. 15

14. A liposomal vector complex as claimed in claim 1, component d) being conjugated to one of the components a) - c) without an anchor, via an anchor or via an anchor lipid as set forth in claim 12 or 13. 20

15. A liposomal vector complex as claimed in claim 1, component d) not being covalently embedded in the liposome surface.

16. A liposomal vector complex as claimed in claims 1-15, the target cell being a tissue cell, an epithelial cell, an endothelial cell, a blood cell, 25 a leukemia cell or a tumor cell.

17. A liposomal vector complex as claimed in claim 1, component e) being the functional sequence of the subunit HA-2 of the hemagglutin of the influenza virus or a synthetic derivative thereof. 30

18. The use of a liposomal vector complex as claimed in one of claims 1-17 for the transduction and transfection of cells in vitro.

19. The use as claimed in claim 18 in the presence of serum. 35

20. The use of a liposomal vector complex as claimed in one of claims 1-17 for the transduction and transfection of cells in vivo. 23

21. A cell comprising a liposomal vector complex as claimed in one of claims 1-17.

22. The use of a liposomal vector complex as claimed in one of claims 5 1-17 or of a cell as claimed in claim 21 for the production of a diagnostic for use in vitro and in vivo.

23. The use of a liposomal vector complex as claimed in one of claims 1-17 or of a cell as claimed in claim 21 for the production of a 10 therapeutic for the prophylaxis or therapy of a disease in vivo and ex vivo.

24. The use of the liposomal vector complex as claimed in claim 23 for administration to the skin, to a mucous membrane, in the lung, on 15 the eye, in a body cavity, in the connective tissue, in the muscle, in an organ or in the blood circulation.

25. A process for the preparation of a liposomal vector complex as claimed in one of claims 1-17, where 20 (1) component a) as in claim 1 is mixed with component b) as in claim 1, (2) the complex resulting from step (1) is inserted into the component c) as in claim 1, the mixing ratio of all components being adjusted such that the net charge. of the resulting overall complex is 25 preferably either cationic or anionic; (3) optional component d) as in claim 1 is inserted into the component c) before or after complex formation; (4) optional component e) as in claim 1 is inserted into the complex resulting from steps (2) and (3) or into component c) before complex 30 formation.

26. A process for the preparation of a liposomal vector complex as claimed in one of claims 1-17, the resulting product being lyophilized. 35

27. A process for the preparation of a liposomal vector complex as claimed in one of claims 1-17, the resulting product being aerosolized.