Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
  • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers

Definitions

• vectors for gene therapy have so far been nucleic acid sequences which are complexed with a non-viral carrier (e.g. cationic lipids or cationic polymers) or inserted into a virus.
• a non-viral carrier e.g. cationic lipids or cationic polymers
• RES reticuloendothelial system
• the causes of rapid elimination are varied. They can be: too large a negative or positive charge, too large a volume or an opsonization of the vector particles by blood proteins.
• viral vectors they can furthermore be the binding of the virus envelope proteins to virus-specific receptors in the organs and / or also antibodies or immune cell-specific for the viruses which bind to the vectors and thereby eliminate them.
• the invention relates to new liposomal vector complexes for gene therapy consisting of the following components:
• Component a) can be an unmodified or modified DNA sequence or an unmodified or modified RNA sequence.
• the nucleotide sequence can perform 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 RNAsen. Examples of such nucleotide sequences and their modifications are in Breaker, Nature Biotechnol. 15: 427, 1997; Gerwik, Critical Reviews in Oncogenesis 8: 93, 1997; Mukhopadhyay et al., Crit. Rev. Oncogen.
• 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, into which a nucleic acid sequence foreign to the virus has been inserted using the methods known to those skilled in the art. Examples of such viruses are RTV, AAV and lentiviruses. Such and further examples are from Vile, Nature Biotechnol.
• Component b) is a cationic carrier which condenses component a) and at the same time has lysosomolytic and / or lysosomotropic and / or lysosomotropic properties.
• component b) is a cationic polymer, for example described by Boussif et al., Proc. Natl. Acad. Be. 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.
• a cationic polymer for example described by Boussif et al., Proc. Natl. Acad. Be. USA 92: 7297, 1995; Kaneda et al., Science 243: 375, 1989; Keown et al., Methods in Enzymology 185: 527, 1990; Baker
• PEI polyethylene imine
• component b) is a polyethyleneimine (PEI), in a further particular one
• 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 approximately 2000 Da on average and was produced as in patent application EP -A 0 905 254.
• Component c) represents any liposome with any composition known to the person skilled in the art.
• this liposome has an anionic charge.
• the lipid and phospholipid composition of the anionic liposome is similar to the composition of a virus envelope.
• the preparation of liposomes with an anionic charge has already been described many times, for example in US Pat. Nos. 4,946,787, 4,245,737, 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.
• the invention furthermore relates to the addition of the liposomal vector complexes according to the invention by adding a component d).
• This component d) represents 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 any.
• the type of lipid can be of any type, but naturally occurring lipids are preferred, as described, 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 described in US Pat. No. 5,662,930.
• Component d) is inserted into the liposome according to the invention (component c) using the method known to the person skilled in the art, for example described in US Pat. Nos. US 5,252,348 and US 5,753,258).
• the invention furthermore relates to the supplementation of the liposomal vector complexes according to the invention by adding a component e).
• This component e) represents the functional sequence of a fusion peptide, preferably from the subunit HA-2 of hemagglutinin from the influenza virus (Wagner et al., Proc. Natl. Acad. Sci. USA 89 (17), 7934-7938), ligands, which facilitates the release of the liposome content 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
• component a) in the 1st step component a) is mixed with component b), the mixing ratio being adjusted so that the net charge of the resulting total complex is preferably either cationic or anionic and subsequent
• step 2 the complex resulting from step (1) is introduced into component c), which component d) may already contain, the
• component d) can optionally also be introduced into component c) subsequently step 2;
• component e) is inserted into the complex resulting from step 2 or 3, or, alternatively, component e) is added to component c) before step 2.
• the liposomal vector complexes resulting from these production 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 accumulate, for example, in the 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). Furthermore, the liposomes according to the invention bind via their component d) to the target cell and transfect them, so that the nucleic acid sequence in the vector complex according to the invention is released in the cell.
• this nucleic acid sequence can develop its effect in the target cell, ie, for example, inhibit the transcription or translation of a specific gene or a specific RNA, or the cell transduce to express the RNA or protein encoded by this nucleic acid sequence.
• the transduction rate through the liposomal vector complexes according to the invention is considerably improved in comparison to the existing technology known to the person skilled in the art and is, for example in cell culture, in over 80% of the cells which carry a receptor for component d) and with the liposomal vector complexes according to the invention be brought into contact.
• the vector complexes according to the invention are therefore preferably suitable for in vitro transduction of cells and for in vivo administration with the aim of prophylaxis or therapy of diseases.
• the invention relates to a liposomal vector complex containing the following components: f) a nucleic acid sequence of any length; g) a cationic carrier which condenses component a) and is lysosomolytic and / or lysosomotropic; 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); wherein in the presence of a fusogenic substance (e) the cationic carrier (b) need not be lysosomolytic and / or lysosomotropic; and in which component a) is preferably a polynucleic acid, component b) is a cationic protein, a cationic polymer, or a combination of both.
• component a) is preferably a poly
• the cationic carrier is protamine sulfate.
• component b) is a cationic polymer, in particular polyethyleneimine (PEI), particularly preferably PEI with a molecular weight of on average 2,000-10,000 Da, very particularly preferably a highly branched or low-branched PEI.
• PEI polyethyleneimine
• component c) is composed of phosphatidylsehn, phosphatidylethanolamine, phosphatidylcholine, anchor lipid and cholesterol, particularly preferred as anchor lipid is an N-carboxyphosphatidylethanolamine, e.g. an N-glutaryl phosphatidylethanotamine; and preferably component d) is conjugated to one of components a) - c) without an anchor, via an anchor or via an anchor lipid.
• anchor lipid is an N-carboxyphosphatidylethanolamine, e.g. an N-glutaryl phosphatidylethanotamine
• Another embodiment of the invention is a liposomal vector complex in which component d) is not covalently embedded in the liposome surface.
• Another embodiment of the invention is a liposomal vector complex whose target cell is a tissue cell, an epithelial cell, an endothelial cell, a blood cell, a leukemia cell or a tumor cell.
• Another embodiment of the invention is a liposomal vector complex whose component e) is the functional sequence from the subunit HA-2 of the 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, wherein serum is preferably used in vitro.
• 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 remedy for the prophylaxis or therapy of a disease in vivo and ex vivo, the administration preferably being based on the Skin, on a mucous membrane, in the lungs, on the eye, in a body cavity, in the connective tissue, in the muscle, in an organ or in the bloodstream.
• Another embodiment of the invention is a method for producing a liposomal vector complex, wherein
• component a) is mixed with component b),
• step (2) the complex resulting from step (1) is introduced into component c), the mixing ratio of all components being adjusted so that the net charge of the resulting overall complex is preferably either cationic or anionic;
• component d) is inserted into component c) before or after complex formation
• component e) is inserted into the complex resulting from step (2) and (3) or to component c) before the complex formation; preferably the resulting product is lyophilized or aerosolized.
• a cyclic peptide was synthesized to improve the targeting of the integrin receptors. It is a CDCRGDCFC peptide (Arap W., Pasqualini, R. and Ruoslahti, E. (1998) Science, 279: 377-380; Pasqualini, R. Koivunen, E. Ruoslathi E., Nature Biotech. (1997 ) 15: 542-546) with an additional arginine at the N-terminal end (FW 1163.35). The terminal amino acid reduces the extent of coupling of the peptide to the active site, 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 ⁇ g / 150 ⁇ l Tris buffer 10 mM pH 7.4 or PBS buffer pH 7.4).
• the liposomes are produced using the film / hydration method.
• Chloroform-dissolved lipids and the lipid anchor are pipetted into a 100 ml round-bottomed flask and the chloroform is drawn off for 15 minutes.
• the flask is immersed in a temperature-controlled water bath at a temperature above the The phase transition temperature of the lipids is 30 ° C. in this special case.
• the film is dried under high vacuum for 15 min.
• the film is then hydrated with buffer (Tris 10 mM, pH 7.4 or PBS pH 7.4, other buffers and pH values are also possible).
• buffer Tris 10 mM, pH 7.4 or PBS pH 7.4, other buffers and pH values are also possible.
• the buffer is added to the flask together with a few small glass spheres and the batch is rolled for 45 min under gassing with N 2.
• the flask is also immersed in the 30 ° C water bath.
• MLV multilamellar liposomes
• 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. 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.
• 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.
• Liposomes The size exclusion chromatography is carried out with a Sephadex G 50 column, eluent is Tris buffer 10 mM, pH 7.4 (other buffers and pH values are also possible).
• the coupling yield is carried out by means of a simultaneous approach with RGD labeled with fluorescent dye (5-DTAF) (Product Information Sheet 5-DTAF, Molecular Probes (MP 00143 08/27/95) “Conjugation with Amine-Reactive Probes”) and is at least included 6 ⁇ g RGD / 1 ⁇ mol PL (calculated for the actual amount of lipid with cholesterol)
• the determination of the coupling efficiency can alternatively be carried out using HPLC (Gyongyossy-Issa et al.
• the Covalent Coupling of Arg-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 with RGD is between 100-150 nm.
• the coupling can be modified according to Bogdanov in order to increase the coupling efficiency.
• the coupling can also be carried out using an anchor lipid using the method described by Weissig.
• the resulting lipid anchor-peptide construct like the lipid, can be used in the production of liposomes.
• Example 2 Production of a liposomal vector complex according to the invention in the sequence plasmid, liposome, protamine sulfate and PEI
• the plasmid was mixed with PEI, which was described by Fischer et al. described method (Pharm. Res. 16, 1273-1279, 1999) was produced, or condensed with Lupasol (BASF, Ludwigshafen, Germany).
• the complex is first formed by combining the negatively charged components plasmid DNA (pGI3, Clontech, Heidelberg, Germany) and liposomes (DLPE, DOPS, cholesterol, N-glutaryl-PE 3: 3: 3: 1). The dilution of the solutions must be taken into account to prevent irreversible precipitate formation. The final volume of all components mentioned is 100 ⁇ l. First the buffer (Tris 10 mM, pH 7.4, other buffers and pH values are also possible) is added and 10 ⁇ g plasmid (10 ⁇ g / 60 ⁇ l) and 40 ⁇ g liposomes (variable, between 1 and 6 ⁇ g / ⁇ l) are combined by simple pipetting. The mixture is vortexed.
• the DNA is then condensed by adding the cationic agent; first, 19.96 ⁇ g of protamine sulfate is added (charge ratio +/- 3.3: 1).
• protamine sulfate is quickly added with an Eppendorf pipette and the mixture is mixed by pipetting up and down 10 times and the complex is coated with the lipids.
• This is followed by the addition of 29.7 ⁇ g (amounts up to 16.2 ⁇ g are also possible) PEI (N / P ratio 20.7, reduction to 7.5 possible), another cationic agent.
• 33 ⁇ l PEI solution 0.9 mg / ml is diluted with 250 ⁇ l ultrapure water and added to the batch in partial steps.
• Example 3 Production of a liposomal vector complex according to the invention in the order plasmid, liposome, and PEI
• Example 2 The batch as described in Example 2 can also be prepared without protamine sulfate.
• the manufacturing steps are identical, except for the addition of 19.96 ⁇ g protamine sulfate. Transfection experiments show almost identical efficiency.
• Example 4 Production of a liposomal vector complex according to the invention in the order plasmid, liposome, fusion peptide and PEI.
• fusion peptide hemagglutinin (HA) derived from the membrane protein of the influenza virus (Wagner et al., Proc. Natl. Acad. Sei. USA 89, 7934- 7938, 1992; Smoes & Slepushkin Gene Therapy 5, 955-964, 1998) were added to the liposomes at the beginning of the complex formation and the mixture was 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 ⁇ g) per batch.
• Example 5 Production of a liposomal vector complex according to the invention in the order plasmid, PEI, liposome, fusion peptide.
• the complex formation can also take place by condensation of the DNA by PEI.
• both substances are first combined (one-time addition or in proportions see above) and waited for 15 minutes.
• the liposomes are then pipetted in and the mixture is pipetted up and down several times (preferably 10 times). This complex is also left to stand for 15 minutes before use.
• the amount of liposomal formulation of plasmid, cationic agent and liposomes required for the transfection can be reduced to 5 ⁇ g plasmid / 3 cm dish. In addition, the volume of the formulation can be reduced.
• the complex is first formed by combining the components plasmid DNA and PEI.
• the dilution of the solutions must be taken into account to prevent irreversible precipitate formation.
• the final volume of the two components mentioned is 245.93 ⁇ l.
• the buffer Tris 10 mM, pH 7.8, other buffers and pH values also possible
• 15 ⁇ g plasmid 15 ⁇ g / 90 ⁇ l
• the DNA is then condensed by adding the cationic agent, 44.55 ⁇ g PEI (N / P ratio 20.7, larger amounts and a reduction to 9.79 ⁇ g are also possible).
• PEI solution 0.9 mg / ml is diluted with 106.43 ⁇ l ultrapure water and added to the batch in partial steps.
• the addition takes place in 1 * 100 ⁇ i and 1 * 55.93 ⁇ l steps, 5 * pipette up and down the batch after the addition and then pipette up and down with 100 ⁇ l volume 5 * , wait 15 min.
• 60 ⁇ g liposomes (variable, between 1 and 6 ⁇ g / ⁇ l) are mixed with 15 ⁇ g HA fusion peptide (the amount can be reduced to 0.1 ⁇ g) and added to the plasmid / PEI complex by simple pipetting.
• the batch is pipetted up and down with 100 ⁇ l volume 10 * .
• the mixture is vortexed.
• Complexes of similar effectiveness are also obtained by simply mixing all of the components mentioned.
• Such a complex has a size of 180-250 nm after production and is used immediately after production for the cell culture 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 order plasmid, PEI, liposome.
• the liposomal formulation can be made without the HA fusion peptide.
• the above-mentioned production instructions are used and only the addition of the HA peptide is omitted.
• Example 7 Preparation of a liposomal vector complex according to the invention in the order plasmid, PEI, liposome, with and without HA peptide, with variable volumes.
• the complex formation takes place first by combining the components plasmid DNA and PEI.
• the dilution of the solutions must be taken into account to prevent irreversible precipitate formation.
• the final volume of the two components mentioned is: * a) 465 ⁇ l, b) 245.93 ⁇ l, c) 196.4 ⁇ l, d) 150 ⁇ l.
• the buffer Tris 10 mM, pH 7.8, other buffers and pH values also possible
• 15 ⁇ g plasmid (15 ⁇ g / 90 ⁇ l or 75 ⁇ l) are added.
• the DNA is then condensed by adding the cationic agent, 44.55 ⁇ g PEI (N / P ratio 20.7, larger amounts and a reduction to 9.79 ⁇ g are also possible).
• the cationic agent 44.55 ⁇ g PEI (N / P ratio 20.7, larger amounts and a reduction to 9.79 ⁇ g are also possible).
• 49.5 ⁇ l PEI solution 0.9 mg / ml is diluted with ultrapure water (325.5, 106.43, 56.9, 25.5 ⁇ i) and added to the mixture in partial steps.
• the addition is carried out in 100 ⁇ l and / or variable (55.9, 75, 106 ⁇ l) steps, 5 * pipette up and down the batch after the addition and then pipette up and down with 100 ⁇ l volume 5 *, wait 15 min.
• liposomes (variable, between 1 and 6 ⁇ g / ⁇ l) are mixed with 15 ⁇ g HA fusion peptide (the amount can be reduced to 0.1 ⁇ g).
• This addition of the HA fusion peptide is optional, it can be omitted without major reduction in transfection.
• the liposomes are added to the plasmid / PEI complex by simple pipetting. To do this, add 100 after the addition Pipette ⁇ l volume 10 * up and down. The mixture is vortexed. Complexes of similar effectiveness are also obtained by simply mixing all of the components mentioned. Such a complex has a size of 180-250 nm after production and is used immediately after production for the cell culture experiments. The complexes are stable in the cell culture medium M199 + 10% FCS used for the transfection (size 300-400 nm).
• Example 8 Preparation of a liposomal vector complex according to the invention in the order plasmid, protamine sulfate, PEI, and liposome.
• the complex is first formed by combining the components plasmid DNA and protamine sulfate.
• the dilution of the solutions must be taken into account to prevent irreversible precipitate formation.
• the final volume of the two components mentioned is: «a) 369 ⁇ l, b) 320 ⁇ l.
• the buffer Tris 10 mM, pH 7.8, other buffers and pH values also possible
• 15 ⁇ g plasmid (15 ⁇ g / 90 ⁇ l) added.
• the DNA is then condensed by adding the protamine sulfate (+/- ratio 3.3, larger or smaller amounts are also possible).
• Protamine sulfate is diluted with ultrapure water (29.94 ⁇ g / 105 ⁇ l) and added to the DNA, pipette 10 * being pipetted up and down. The complex is left to mature for 15 minutes. Then 49.5 ⁇ l PEI solution 0.9 mg / ml is diluted with ultrapure water (106.43, 56.9 ⁇ l) and added to the batch in partial steps. The addition takes place in 100 ⁇ l and / or 55.9 ⁇ l steps, pipette 5 * up and down the batch after the addition and then pipette up and down with 100 ⁇ l volume 5 * , wait 15 min.
• liposomes (variable, between 1 and 6 ⁇ g / ⁇ i) are mixed with 15 ⁇ g HA fusion peptide (the amount can be reduced to 0.1 ⁇ g).
• This addition of the HA fusion peptide is optional, it can be omitted without major reduction in transfection.
• the liposomes are added to the plasmid / PS / PEI complex by simple pipetting. For this purpose, the batch is pipetted up and down with 100 ⁇ l volume 10 *. The mixture is vortexed. Complexes of similar effectiveness are also obtained by simply mixing all of the components mentioned. Such a complex has a size of after production 180-300 nm and is used for cell culture experiments immediately after production.
• 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 different charge ratios, whereby both the PS and the PEI content can be varied. The volumes can also be selected to be larger or smaller.
• Example 9 Production of a liposomal vector complex according to the invention in the order plasmid, protamine sulfate, PEI, and liposome with a reduced PEI content.
• the complex is first formed by combining the components plasmid DNA and protamine sulfate.
• the dilution of the solutions must be taken into account to prevent irreversible precipitate formation.
• the final volume of the two components mentioned is: «319 ⁇ l.
• the buffer Tris 10 mM, pH 7.8, other buffers and pH values also possible
• 15 ⁇ g plasmid (15 ⁇ g / 90 ⁇ l) added.
• the DNA is then condensed by adding the protamine sulfate (+/- ratio 3.3, larger or smaller amounts are also possible).
• Protamine sulfate is diluted with ultrapure water (29.94 ⁇ g / 105 ⁇ l) and added to the DNA, pipette 10 * being pipetted up and down. The complex is left to mature for 15 minutes. Then 30 ⁇ l (N / P ratio 12.5) or 18 ⁇ l (N / P ratio 7.5) PEI solution 0.9 mg / ml are diluted with ultrapure water to 106 ⁇ l (76 or 88 ⁇ l) and added to the batch in partial steps. The addition is carried out in 106 ⁇ l steps, 10 * the batch after the addition and pipette off, wait 15 min.
• liposomes (variable, between 1 and 6 ⁇ g / ⁇ l) are mixed with 15 ⁇ g HA fusion peptide (the amount can be reduced to 0.1 ⁇ g).
• This addition of the HA fusion peptide is optional, it can be omitted without major reduction in transfection.
• the liposomes are added to the plasmid / PS / PEI complex by simple pipetting. For this purpose, the batch is pipetted up and down with 100 ⁇ l volume 10 * . The mixture is vortexed. Complexes of similar effectiveness are also obtained by simply mixing all of the components mentioned.
• Example 10 Preparation of a liposomal vector complex according to the invention in the order plasmid, PEI, protamine sulfate, and liposome.
• the complex is first formed by combining the components plasmid DNA and PEI.
• the dilution of the solutions must be taken into account to prevent irreversible precipitate formation.
• the final volume of the two components mentioned is: «246 ⁇ l.
• the buffer Tris 10 mM, pH 7.8, other buffers and pH values also possible
• 15 ⁇ g plasmid 15 ⁇ g / 90 ⁇ l
• the DNA is then condensed by adding 44.55 ⁇ g PEI (N / P ratio 20.7, larger quantities and a reduction is also possible).
• PEI solution 0.9 mg / ml is diluted with ultrapure water (to 155.93 ⁇ l) and added to the batch in partial steps.
• the addition is carried out in 100 ul and 55.9 .mu.l steps with 5 * the batch after addition of pipetting up and down and * and on-pipetting finally with 100 ul volumes of 5, 15 min wait.
• the protamine sulfate is added (+/- ratio 3.3, larger or smaller amounts are also possible).
• Protamine sulfate is diluted with ultrapure water (29.94 ⁇ g / 105 ⁇ l) and added to the DNA, pipette 10 * being pipetted up and down. The complex is left to mature for 15 minutes.
• liposomes (variable, between 1 and 6 ⁇ g / ⁇ l) are mixed with 15 ⁇ g HA fusion peptide (the amount can be reduced to 0.1 ⁇ g).
• This addition of the HA fusion peptide is optional, it can be omitted without major reduction in transfection.
• the liposomes are added to the plasmid / PEI / PS complex by simple pipetting. For this purpose, the batch is pipetted up and down with 100 ⁇ l volume 10 * . The mixture is vortexed. Complexes of similar effectiveness are also obtained by simply mixing all of the components mentioned.
• Example 11 Preparation of a liposomal vector complex according to the invention in the order plasmid, PEI, protamine sulfate, and liposome with a reduced PEI content.
• the complex is first formed by combining the components plasmid DNA and PEI. The dilution of the solutions must be taken into account to prevent irreversible precipitate formation. Depending on the batch type, the final volume of the two components mentioned is: «196 ⁇ l. First, the buffer (Tris 10 mM, pH 7.8, other buffers and pH values also possible) is introduced and 15 ⁇ g plasmid (15 ⁇ g / 90 ⁇ l) added.
• the DNA is then condensed by adding 9.75 ⁇ g, 16.2 ⁇ g or 27 ⁇ g PEI (N / P ratio 4.5, 7.5, 12.5, larger amounts and a reduction are also possible, for example N / P ratio 1.8).
• 10.86 ⁇ l, 18 ⁇ l or 30 ⁇ l PEI solution 0.9 mg / ml is diluted with ultrapure water (to 106 ⁇ l) and added to the mixture with pipetting up and down 10 times.
• protamine sulfate is added (+/- ratio 3.3, larger or smaller amounts are also possible).
• Protamine sulfate is diluted with ultrapure water (29.94 ⁇ g / 105 ⁇ l) and added to the DNA / PEI complex, pipetting 10 * up and down. The complex is left to mature for 15 minutes.
• 60 ⁇ g liposomes (variable, between 1 and 6 ⁇ g / ⁇ l) are mixed with 15 ⁇ g HA fusion peptide (the amount can be reduced to 0.1 ⁇ g).
• This addition of the HA fusion peptide is optional, it can be omitted without major reduction in transfection.
• the liposomes are added to the plasmid / PEI / PS complex by simple pipetting.
• the batch is pipetted up and down with 100 ⁇ l volume 10 *.
• the mixture is vortexed.
• Complexes of similar effectiveness are also obtained by simply mixing all of the components mentioned.
• Example 12 Production of a liposomal vector complex according to the invention in the order plasmid, PEI, and liposome with an increased and reduced lipid content.
• the complex is first formed by combining the components plasmid DNA and PEI.
• the dilution of the solutions must be taken into account to prevent irreversible precipitate formation.
• the final volume of the two components mentioned is 245.93 ⁇ l.
• First the buffer (Tris 10 mM, pH 7.8, other buffers and pH values also possible) is placed in front and 15 ⁇ g plasmid (15 ⁇ g / 90 ⁇ l) added.
• the DNA is then condensed by adding the cationic agent, 44.55 ⁇ g PEI (N / P ratio 20.7, larger amounts and a reduction is also possible).
• 49.5 ⁇ l PEI solution 0.9 mg / ml is diluted with 106.43 ⁇ l ultrapure water and added to the batch in partial steps.
• the addition takes place in 1 * 100 ⁇ l and 1 * 55.93 ⁇ l steps, 5 * pipette up and down the batch after the addition and then pipette up and down with 100 ⁇ l volume 5 *, wait 15 min. 75, 45 and 30 ⁇ g liposomes (between 1 and 6 ⁇ g / ⁇ l) are added to the plasmid / PEI complex by simple pipetting.
• the batch is pipetted up and down with 100 ⁇ l volume 10 * .
• the proportion of liposomes used can also be increased significantly to 10 times the amount.
• Example 13 Biological testing of the liposomal vector complexes according to the invention in different cell cultures
• the complex is first formed by combining the components plasmid DNA and PEI.
• the dilution of the solutions must be taken into account to prevent irreversible precipitate formation.
• the final volume of the two components mentioned is 245.93 ⁇ l.
• the buffer Tris 10 mM, pH 7.8, other buffers and pH values also possible
• 15 ⁇ g plasmid 15 ⁇ g / 90 ⁇ l
• the DNA is then condensed by adding the cationic agent, 44.55 ⁇ g PEI (N / P ratio 20.7, larger amounts and a reduction is also possible).
• 49.5 ⁇ l PEI solution 0.9 mg / ml is diluted with 106.43 ⁇ l ultrapure water and added to the batch in partial steps.
• the addition takes place in 1 * 100 ⁇ l and 1 * 55.93 ⁇ l steps, 5 * pipette up and down the batch after the addition and then pipette up and down with 100 ⁇ l volume 5 *, wait 15 min. 60 ⁇ g liposomes (between 1 and 6 ⁇ g / ⁇ l) with and without a coupled RGD targetor (RGD binds to the ⁇ v ß 3 receptor) are added to the plasmid / PEI complex by simple pipetting. For this purpose, the batch is pipetted up and down with 100 ⁇ l volume 10 *. The mixture is divided into 3 aliquots and an aliquot is placed on a 3 cm dish. The triplicate was placed on 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 ° C. for 1-6 hours. The cells were subsequently washed and incubated in the fresh cell culture medium for a further 24-48 hours. The successful uptake of the complexes into the cell, the transcription, and the expression of the reporter gene in the plasmid were then measured by detecting the GFP autofluorescence, luciferase assay, and FACS analysis. Results of the FACS analysis are summarized in the following table.

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