CA2375854A1 - Novel liposomal vector complexes and their use in gene therapy - Google Patents
Novel liposomal vector complexes and their use in gene therapy Download PDFInfo
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- A61K9/10—Dispersions; Emulsions
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Abstract
The invention relates to a novel liposomal vector complex, containing the following components: a) a nucleic acid sequence of any length; b) a cationi c 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
29/11 '0l 14:54 FAX ++49 89 92805444 BARDEftLE OFFICE -~ GOUDREAU 0]024 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.
cationic lipids or cationic polymers) or are inserted into a virus.
Previous experience with such vectors in the gene therapy of ail 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 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 a1. Gene Ther. 6: 595, 1999; Dash et al., Gene Ther.
t 5 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 '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 opsoni2ation 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.
Previous experience additionally shows that the coupling or insertion of a target cell-specific figand into the vector complex does not significantly decrease its rapid elimination after administration into the blood circulation.
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 tong as possible in the circulation and are not prematurely eliminated from the circulation. In order to decrease the elimination of cationic lipids or 29/11. '0l 14:55 FAX ++48 89 92805444 BARDEHLE OFFICE -~ GOUDREAU I~J025 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., FEES Lett 284; 263, 1991; Ogris et al., Gene Ther. 6: 595, 1999), vinyl polymers (Torchilin et al., Biochim. Biophys. Res.
Acta 1195: 181, 1994) or other amphipathic polymers (Woodle et al., Bioconjugat. Chem. 5: 493, i 994) 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 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; Baiicki 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, i 998).
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 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 Ther. 6: 595, 1999).
At the same time, however, it was also possible in the lung, spleen and liver to detect va considerable concentration of the vectors and transduction of the tissue cells in these organs (Ogris et al., Gene Ther. 8: 595, 1999), so that it can gibe concluded that, for example, the coupling of PEG does bring about arp improvement, but still no optimization of the distribution of vectors.
General description of the invention 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;
29/11 'O1 14:56 FAX ++49 89 92805444 BARDEHL.E OFFICE -~ GOiJDREAU C~J026 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 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 lysosornotropic.
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 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, 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 nucleic acid sequence foreign to the virus has been inserted using the methods known to the person skiNed 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.
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 lysosomolyticaily and/or lysosomotropically and/or iysosomotropic properties.
According to this invention, in a particular embodiment 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 29/1.1 'O1 14:56 FAX ++49 89 92805444 BARDEALE OFFICE -. GOLTDREAiJ f~/027 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 ai., Human Gene Ther. 7: 2123, 1996 and Solodin et al., Biochem. 34: 13537, 1995. The polyethyleneimine (PEI} described by Boussif, when used as a 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 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 application EP-A 0 905 254. High-branched-chain PEI (LupasolC, 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.
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 liposornes having anionic 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, 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.
The preparation of liposomes which are similar to virus coats was described, for example, in the US patents Nos. US 5,252,348; US
cationic lipids or cationic polymers) or are inserted into a virus.
Previous experience with such vectors in the gene therapy of ail 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 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 a1. Gene Ther. 6: 595, 1999; Dash et al., Gene Ther.
t 5 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 '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 opsoni2ation 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.
Previous experience additionally shows that the coupling or insertion of a target cell-specific figand into the vector complex does not significantly decrease its rapid elimination after administration into the blood circulation.
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 tong as possible in the circulation and are not prematurely eliminated from the circulation. In order to decrease the elimination of cationic lipids or 29/11. '0l 14:55 FAX ++48 89 92805444 BARDEHLE OFFICE -~ GOUDREAU I~J025 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., FEES Lett 284; 263, 1991; Ogris et al., Gene Ther. 6: 595, 1999), vinyl polymers (Torchilin et al., Biochim. Biophys. Res.
Acta 1195: 181, 1994) or other amphipathic polymers (Woodle et al., Bioconjugat. Chem. 5: 493, i 994) 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 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; Baiicki 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, i 998).
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 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 Ther. 6: 595, 1999).
At the same time, however, it was also possible in the lung, spleen and liver to detect va considerable concentration of the vectors and transduction of the tissue cells in these organs (Ogris et al., Gene Ther. 8: 595, 1999), so that it can gibe concluded that, for example, the coupling of PEG does bring about arp improvement, but still no optimization of the distribution of vectors.
General description of the invention 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;
29/11 'O1 14:56 FAX ++49 89 92805444 BARDEHL.E OFFICE -~ GOiJDREAU C~J026 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 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 lysosornotropic.
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 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, 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 nucleic acid sequence foreign to the virus has been inserted using the methods known to the person skiNed 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.
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 lysosomolyticaily and/or lysosomotropically and/or iysosomotropic properties.
According to this invention, in a particular embodiment 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 29/1.1 'O1 14:56 FAX ++49 89 92805444 BARDEALE OFFICE -. GOLTDREAiJ f~/027 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 ai., Human Gene Ther. 7: 2123, 1996 and Solodin et al., Biochem. 34: 13537, 1995. The polyethyleneimine (PEI} described by Boussif, when used as a 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 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 application EP-A 0 905 254. High-branched-chain PEI (LupasolC, 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.
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 liposornes having anionic 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, 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.
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.
The invention additionally relates to the completion of the liposornal vector complexes according to the invention by addition of a component d).
29/11 'O1 14:57 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU I~J028 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 arbi#rary.
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 - 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 unpublished).
The type of lipid can be arbitrary, but naturally occurring lipids, such as described, far 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.
The conjugation of lipids to the target cell-specific tigand 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.
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).
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. Natl. Acad. Sci. USA 89{17), 7934-7938) ligands, which facilitate 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 29/11 '0l 14:57 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU 0]029 carried out using methods known to the person skilled in the art, for example in such a way that - in the 1s~ step component a) is mixed with component b}, the mixing ratio being adjusted such that the net charge of the resulting total complex is preferably either cationic or anionic and subsequently - in the 2"d step the complex resulting from step (1} is inserted into the component c}, which can already contain component d), the mixing 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~d step component d) can also optionally be inserted into component c) following step 2; and - in a 4t" 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.
The liposomal vector complexes resulting from these preparation steps have a diameter of 100 - 600 nm and a cationic or anionic ctlarge, preferably a diameter of 100 - 300 nm and an anionic charge.
The liposomes according to the invention are enriched, for example, in the tumor vascular bed (Unezaki et al., Int. J. Pharmac. 174:11, 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 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 the expression of the RNA ar of the protein encoded by this nucleic acid sequence.
The iransduction rate by the liposomal vector complexes according to the invention is considerably improved in comparison to the existing technique 29/11 'O1 14:58 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOUDREAU f~ 030 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.
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.
The invention relates to a liposomai 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;
h) lipids and phospholipids which form a liposome;
t) optionally a ligand which has a binding site for a target cell;
j) optionally a fusogenic substance which can replace the lysosomolytic and/or lysasomotropic function of component b);
where in the presence of a fusogenic substances) the cationic carrier {b) 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 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 - t 0,000 Da, very 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-carboxyphosphatidylethanolamine, e.g. an N-gtutarylphosphatidylethanol-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.
29/11 'O1 14:58 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOiTDREAU C~J031 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 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 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.
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 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 {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 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 formation;
the resulting product preferably being lyophilized or aerosolized.
Examples for illustration of the concept of the invention 29/11. '0l 14:59 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOLTDREAU I~j032 The following examples are intended to illustrate how the present invention could be carried out.
Example 1:
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, 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.
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 ~g/150 ~I of tris buffer 10 mM pH 7.4 or PBS buffer pH 7.4) before use.
Preparation of the liposomes Material Substance Manufacturer Batch Parts FW
DAPS Avanti 181 PS-P36a 3 810 Sodium salt DLPE S ena 0998 3 579.76 CholesterolCalbiochem 228111 3 386.7 N-glut-PE in-house synthesis16118 1 805.97 or Avanti 29/11 'O1 14:59 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU f~J033 . 10 Stock solutions Substance ConcentrationBatch Vol. Amount of [p,mol/ml] employed substance [~I] employed [~mol]
DOPS 16.13 15128 i 86 3 DLPE 33.47 09039 89.7 3 Cholesterol24.95 09039 120.2 3 N- It-PE 2 16118 500 1 The liposomes are prepared by the film/hydration method. The lipids 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°C. For the complete removal of the 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 min with N2 aeration, the flask also dips into the warm water bath at 30°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 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 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 carried out under an air hood in a sterile Eppendorf cap.
29/7.1 'O1 15:00 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOUDREAU f~034 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 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. FEES Lett. 202, 1986, 86-90; Bogdanov et al., "Protein immobilization on the surface of liposomes via carbodiimide 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, 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 Substance Conc. Batch Solvent Amount Amount of mark employed substance em to ed Liposomes 10 pmoi/ml12039 Iris 400 w1 4 ~cmol buffer H 7.4 EDAC pure 25H0993, solid 3.5 mg 18.26 substanceSi ma pmol RGD 250 fig/ 22049 tris 150 fc( 0.215 buffer =
solution 150 ~1 H 7.4 250 ~c umol Firstly, the carboxyl group of the giutaric acid radical of the lipid anchor is activated by the addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide [3, p. 170]. For this, 400 ~l of the liposome suspension (4 pmol of total lipid, 0.4 ~mol 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 formation of an amide. 250 ~g of RGD dissolved in PBS buffer pH 7.4 or 29/11 '01 15:01 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU f~J035 tris buffer 10 mM pH 7.4 (250 ~eg/150 p1, 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 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 information sheet 5-DTAF, Molecular Probes (MP 00143 08/27/95) "Conjugation with Amine-Reactive Probes") and is at least 6 ~g of RGD/
1 Imo! 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-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 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 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, 7 273-1279, 1999), or using Lupasol (BASF, Ludwigshafen, Germany).
Complex formation is firstly carried out by mixing together the negatively charged constituents plasmid DNA (pGl3, Clontech, Heidelberg, Germany) and liposomes (DLPE, D(JPS, 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 prevent irreversible precipitate formation. The final volume of all constituents mentioned is 100 ~I. Firstly, the buffer (iris 10 mM, pH 7.4, other buffers and pHs are likewise possible) is introduced and 10 ~g of plasmid (10 ~cg/60 p1) and also 40 ~cg of Jiposomes (variable, between 1 and 6 pg/pl) are mixed together by simple pipetting. The mixture is vortexed.
29/11 'O1 15:01 FAX ++49 89 92805444 BARDEHLE OFFICE -. GOUDREAU C~03B
The condensation of the DNA is then carried out by addition of the cationic agent, firstly 19.96 ~g of protamine sulfate is added (charge ratio +I-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 coating of the complex with the lipids is achieved. 29.7 beg (amounts up to 16.2 ~g are likewise possible) of PEI (NIP ratio 20.7, reduction to 7.5 possible), a further cationic agent, are then added. For this, 33 p.1 of PEI
solution 0.9 mg/ml are diluted with 250 ~,l of high-purity water and added to the batch in substeps. The addition is carried out in 2 x 100 ~! and 1 x 85 ~I
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).
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 protamine sulfate. The preparation steps are identical, with the exception of the addition of 19.96 ug of protamine sulfate. Transfection experiments show almost identical efficiency.
example 4: Preparation of a liposomal vector complex according to the irhvention in the sequence plasmid, liposome, fusion peptide, and PEI
T'he 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., 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 iiposomes according to Example 3. The concentration of the WA peptide can be between 0.1 and 1 nmol (1 - 10 fig) per batch.
Example 5: Preparation of a liposomal vector complex according to the invention in the sequence plasmid, PEI, liposome, fusion peptide.
29/11 ' O1. 15: 02 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAIT f~ 037 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).
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 pg of plasmid/3 cm dish. In addition, the volume of the formulation can be decreased.
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 ~.1. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs are likewise possible) is introduced and 15 ug of plasmid (15 ~.g/90 ~!) are added thereto. The condensation of the DNA is then carried out by addition of the cationic agent, of 44.55 ~g of PEI (N!P ratio 20.7, larger amounts and a reduction to 9.79 ug are likewise possible). For this, 49.5 p1 of PEI solution 0.9 mg/ml are diluted with 106.43 y1 of high-purity water and added to the batch in substeps. The addition is carried out in 1 x 100 ~I and 1 x 55.93 u1 steps, the batch is pipetted to and fro 5 x after the addition and finally pipetted to and fro 5 x with a 100 u1 volume, wait for 15 min. 60 ~g of liposomes (variable, between 1 and 6 ~cg/~I) are mixed with 15 ~g of HA fusion peptide (reduction of the amount to 0.1 ~cg possible) and added to the plasmidIPEI
complex by simple pipetting. For this, the batch is pipetted to and fro 10 x with a 100 ~I 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 experiments. The complexes are stable in the cell culture medium M 199 +
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, PEf, liposome 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.
29/11 '01. 15:02 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU f~038 _ 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.
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 fins! volume of the two constituents mentioned is, depending on the batch type: = a) 465 ~I, b) 245.93 ~I, c) 196.4 ~I, d} 150 p1. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs are likewise possible) is introduced and 15 ~g of plasmid (15 ~g/90 ~l or 75 u1) added thereto. The condensation of the DNA is then carried out by addition of the cationic agent, of 44.55 beg of PEI (N/P ratio 20.7, larger amounts and a reduction to 9.79 fxg are likewise possible). For this, 49.5 u! of PEI solution 0.9 mg/ml are diluted with high-purity water (325.5, 106.43, 5fi.9, 25.5 ~I) and added to the batch in substeps. The addition is carried out in 100 ;~I and/or variable (55.9, 75, 106 u1) 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 ~l volume, wait for 15 min. so ug of liposomes (variable, between 1 and 6 ~gipl) are mixed with 15 pg of HA fusion peptide (reduction of the amount to 0.1 ~g 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 u1 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 180-250 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 300-400 nm).
Example 8: Preparation of a Iiposomal vector complex according to the invention in the sequence plasmid, protamine sulfate, ~PEl~and liposome.
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 p1, b) 320 ~I. Firstly, the buffer (tris 29/1.1 'O1 15:03 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOUDREAU ~ 039 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and p.g of plasmid (15 ug/90 ~I) 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 pg/105 ~I) 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 -~I of PEI solution 0.9 mg/ml are then diluted with high-purity water (106.43, 56.9 1u1) and added to the batch in substeps. The addition is carried out in 700 p! and/or 55.9 p1 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 p1 volume, wait for 15 min. 60 pg of liposomes (variable, between 1 and 6 ~g/~I) 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 u1 volume. The mixture is vortexed. Complexes of similar activity are also obtained by simple mixing of ail components indicated. After preparation, a complex of this type has a size of 180-300 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 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. !n this case, larger or smaller volumes can likewise be chosen.
Example 9: Preparation of a liposomai vector complex according to the invention in the sequence plasmid, protamine sulfate, PEI and liposome having a reduced PEI content.
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: = 319 p1. Firstly, the buffer (tris 10 mM, pH
7.8, olher buffers and pHs likewise possible) is introduced and 15 ~,g of plasmid {15 pgI90 ~I) are added. The condensation of the DNA is then carried out by addition of the protamine sulfate (+I- ratio 3.3, larger or smaller amounts are likewise possible). Protamine sulfate is diluted with 29/1.1 ' Ol 1.5 : 04 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOiIDREAI1 l~J
_ '! 7 high-purity water (29.94 pg/105 ~I) and added to the DNA, the batch being pipetted to and fro 10 x. The complex is allowed to stand for t 5 min for maturation. 30 ~! (NIP ratio 12.5) or 18 ~I (NIP ratio 7.5) of PEI solution 0.9 mg/ml are then diluted to 106 ~I with high-purity water (76 or 88 pf) and added to the batch in substeps. The addition is carried out in 106 p1 steps, after the addition pipette the batch to and fro 10 x each, wait for 15 min. 60 pg of liposomes (variable, between 1 and 6 pgl~l) are mixed with 15 ~g of HA fusion peptide (reduction of the amount to 0.1 ~.g 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/PSIPEI complex by simple pipetting. For this, the batch is pipetted to and fro 10 x with a 100 p1 volume after addition. The mixture is vortexed.
Complexes of similar activity are also obtained by simple mixing of all components indicated.
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 togefher the constituents plasmid DNA and PEI. !n 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 Vii. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and 15 ~g of plasmid (15 pg/90 ~~I) are added thereto. The condensation of the DNA is Then carried out by addition of 44.55 pg of PEI (NIP ratio 20.7, larger amounts and a reduction are likewise possible). For this, 49.5 p1 of PEI solution 0.9 mg/ml are diluted with high-purity water (to 155.93 p1) and added to the batch in substeps.
The addition is carried out in 100 ~I and 55.9 ,~l 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 p1 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 ~g/105 ~I) 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. 60 ug of liposomes (variable, between 1 and 6 ~,g/~I) are mixed with 15 ~g of HA fusion peptide (reduction of the amount to 0.1 ~g 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.
29/11 'O1 15:05 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOUDREAU C~j041 For this, the batch is pipetted to and fro i0 x after the addition with a 100 ~I
volume. The mixture is vortexed. Complexes of similar activity are also obtained by simple mixing of all components indicated.
Example 11: Preparation of a iiposomal vector complex according to the invention in the sequence plasmid, PE1, protamine sulfate and liposome having a reduced PEI 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 final volume of the two constituents mentioned is, depending on the batch type: = 196 u1. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible} is introduced and 15 pg of plasmid (15 ug/90 yi) are added. The condensation of the DNA is then carried out by addition of 9.75 fig, 16.2 ~g or 27 ~g 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 ~I
18 ~I or 30 ~l of PEI solution 0.9 mg/ml are diluted with high-purity water (to 106 pI) and added to the batch while pipetting to and fro 10 x. The 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 ~g1105 ~.I) 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 pg of liposomes (variable, between 1 and 6 ~g/~,I) are mixed with 15 ~g of HA fusion peptide (reduction of the amount to O.i ~g 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 ~l volume.
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 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 29/11 'O1 15:08 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOLTDREALT f~j042 i9 final volume of the two constituents mentioned is 245.93 ~l. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and 15 pg of plasmid (15 pgI90 p1) are added thereto. The condensation of the DNA is then carried out by addition of the cationic agent, 44.55 pg of PEl (NIP ratio 20.7, larger amounts and a reduction are likewise possible). For this, 49.5 p1 of PEI solution 0.9 mglml are diluted with 106.43 ~l of high-purity water and added to the batch in subsieps. The addition is carried out in 1 x 100 fd and 1 x 55.93 ~l 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 ~d volume, wait for 15 min. 75, 45 and 30 ~g of liposomes (between 1 and 6 pglpl) are added to the plasmidIPEI complex by simple pipetting. For this, the batch is pipetted to and fro 10 x after the addition with a 100 p1 volume. The content of the liposomes used can moreover be markedly increased to 10 x the amount.
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 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 p1. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and 15 ~g of plasmid (15 ug/90 ~l) are added thereto. The condensation of the DNA is then carried out by addition of the cationic agent, 44.55 pg PEl (NIP ratio 20.7, larger amounts and a reduction are likewise possible). For this, 49.5 ~1 of PEI solution 0.9 mg/rnf are diluted with 106.43 w1 of high-purity water and added to the batch in substeps. The addition is carried out in 1 x 100 p! and 1 x 55.93 ~I steps, after the addition the batch is pipetted to and fro 5 x and finally pipetted to and fro 5 x with a 100 u1 volume, wait for 15 min. 60 ~g of liposomes (between 1 and 6 pg/~l) with and without coupled RGD targeter (RGD binds to the a"~ 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 p1 volume.
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 FRCS analysi .
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.
29/11 'O1 ---15:06 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU ~ 043 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 defection of the GFP autofluorescence, 5 luciferase assay, and FACS analysis were then measured. Results of the FACS analysis are compiled in the following table.
f Cells + RGD - RGD
HUVEC rima 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 rostate carcinoma cells 1 1 pC3 rostate carcinoma cells 95 21 HeLa cervical carcinoma cells 4 33 LoVo colon carcinoma cells 8 1 A549 lun carcinoma cells 23 11 MCF-7 breast cancer cells ~3 34 ' JEG-3 chorionic carcinoma cells ~4
The invention additionally relates to the completion of the liposornal vector complexes according to the invention by addition of a component d).
29/11 'O1 14:57 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU I~J028 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 arbi#rary.
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 - 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 unpublished).
The type of lipid can be arbitrary, but naturally occurring lipids, such as described, far 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.
The conjugation of lipids to the target cell-specific tigand 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.
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).
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. Natl. Acad. Sci. USA 89{17), 7934-7938) ligands, which facilitate 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 29/11 '0l 14:57 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU 0]029 carried out using methods known to the person skilled in the art, for example in such a way that - in the 1s~ step component a) is mixed with component b}, the mixing ratio being adjusted such that the net charge of the resulting total complex is preferably either cationic or anionic and subsequently - in the 2"d step the complex resulting from step (1} is inserted into the component c}, which can already contain component d), the mixing 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~d step component d) can also optionally be inserted into component c) following step 2; and - in a 4t" 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.
The liposomal vector complexes resulting from these preparation steps have a diameter of 100 - 600 nm and a cationic or anionic ctlarge, preferably a diameter of 100 - 300 nm and an anionic charge.
The liposomes according to the invention are enriched, for example, in the tumor vascular bed (Unezaki et al., Int. J. Pharmac. 174:11, 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 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 the expression of the RNA ar of the protein encoded by this nucleic acid sequence.
The iransduction rate by the liposomal vector complexes according to the invention is considerably improved in comparison to the existing technique 29/11 'O1 14:58 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOUDREAU f~ 030 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.
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.
The invention relates to a liposomai 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;
h) lipids and phospholipids which form a liposome;
t) optionally a ligand which has a binding site for a target cell;
j) optionally a fusogenic substance which can replace the lysosomolytic and/or lysasomotropic function of component b);
where in the presence of a fusogenic substances) the cationic carrier {b) 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 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 - t 0,000 Da, very 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-carboxyphosphatidylethanolamine, e.g. an N-gtutarylphosphatidylethanol-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.
29/11 'O1 14:58 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOiTDREAU C~J031 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 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 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.
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 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 {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 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 formation;
the resulting product preferably being lyophilized or aerosolized.
Examples for illustration of the concept of the invention 29/11. '0l 14:59 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOLTDREAU I~j032 The following examples are intended to illustrate how the present invention could be carried out.
Example 1:
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, 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.
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 ~g/150 ~I of tris buffer 10 mM pH 7.4 or PBS buffer pH 7.4) before use.
Preparation of the liposomes Material Substance Manufacturer Batch Parts FW
DAPS Avanti 181 PS-P36a 3 810 Sodium salt DLPE S ena 0998 3 579.76 CholesterolCalbiochem 228111 3 386.7 N-glut-PE in-house synthesis16118 1 805.97 or Avanti 29/11 'O1 14:59 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU f~J033 . 10 Stock solutions Substance ConcentrationBatch Vol. Amount of [p,mol/ml] employed substance [~I] employed [~mol]
DOPS 16.13 15128 i 86 3 DLPE 33.47 09039 89.7 3 Cholesterol24.95 09039 120.2 3 N- It-PE 2 16118 500 1 The liposomes are prepared by the film/hydration method. The lipids 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°C. For the complete removal of the 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 min with N2 aeration, the flask also dips into the warm water bath at 30°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 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 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 carried out under an air hood in a sterile Eppendorf cap.
29/7.1 'O1 15:00 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOUDREAU f~034 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 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. FEES Lett. 202, 1986, 86-90; Bogdanov et al., "Protein immobilization on the surface of liposomes via carbodiimide 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, 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 Substance Conc. Batch Solvent Amount Amount of mark employed substance em to ed Liposomes 10 pmoi/ml12039 Iris 400 w1 4 ~cmol buffer H 7.4 EDAC pure 25H0993, solid 3.5 mg 18.26 substanceSi ma pmol RGD 250 fig/ 22049 tris 150 fc( 0.215 buffer =
solution 150 ~1 H 7.4 250 ~c umol Firstly, the carboxyl group of the giutaric acid radical of the lipid anchor is activated by the addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide [3, p. 170]. For this, 400 ~l of the liposome suspension (4 pmol of total lipid, 0.4 ~mol 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 formation of an amide. 250 ~g of RGD dissolved in PBS buffer pH 7.4 or 29/11 '01 15:01 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU f~J035 tris buffer 10 mM pH 7.4 (250 ~eg/150 p1, 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 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 information sheet 5-DTAF, Molecular Probes (MP 00143 08/27/95) "Conjugation with Amine-Reactive Probes") and is at least 6 ~g of RGD/
1 Imo! 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-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 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 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, 7 273-1279, 1999), or using Lupasol (BASF, Ludwigshafen, Germany).
Complex formation is firstly carried out by mixing together the negatively charged constituents plasmid DNA (pGl3, Clontech, Heidelberg, Germany) and liposomes (DLPE, D(JPS, 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 prevent irreversible precipitate formation. The final volume of all constituents mentioned is 100 ~I. Firstly, the buffer (iris 10 mM, pH 7.4, other buffers and pHs are likewise possible) is introduced and 10 ~g of plasmid (10 ~cg/60 p1) and also 40 ~cg of Jiposomes (variable, between 1 and 6 pg/pl) are mixed together by simple pipetting. The mixture is vortexed.
29/11 'O1 15:01 FAX ++49 89 92805444 BARDEHLE OFFICE -. GOUDREAU C~03B
The condensation of the DNA is then carried out by addition of the cationic agent, firstly 19.96 ~g of protamine sulfate is added (charge ratio +I-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 coating of the complex with the lipids is achieved. 29.7 beg (amounts up to 16.2 ~g are likewise possible) of PEI (NIP ratio 20.7, reduction to 7.5 possible), a further cationic agent, are then added. For this, 33 p.1 of PEI
solution 0.9 mg/ml are diluted with 250 ~,l of high-purity water and added to the batch in substeps. The addition is carried out in 2 x 100 ~! and 1 x 85 ~I
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).
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 protamine sulfate. The preparation steps are identical, with the exception of the addition of 19.96 ug of protamine sulfate. Transfection experiments show almost identical efficiency.
example 4: Preparation of a liposomal vector complex according to the irhvention in the sequence plasmid, liposome, fusion peptide, and PEI
T'he 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., 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 iiposomes according to Example 3. The concentration of the WA peptide can be between 0.1 and 1 nmol (1 - 10 fig) per batch.
Example 5: Preparation of a liposomal vector complex according to the invention in the sequence plasmid, PEI, liposome, fusion peptide.
29/11 ' O1. 15: 02 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAIT f~ 037 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).
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 pg of plasmid/3 cm dish. In addition, the volume of the formulation can be decreased.
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 ~.1. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs are likewise possible) is introduced and 15 ug of plasmid (15 ~.g/90 ~!) are added thereto. The condensation of the DNA is then carried out by addition of the cationic agent, of 44.55 ~g of PEI (N!P ratio 20.7, larger amounts and a reduction to 9.79 ug are likewise possible). For this, 49.5 p1 of PEI solution 0.9 mg/ml are diluted with 106.43 y1 of high-purity water and added to the batch in substeps. The addition is carried out in 1 x 100 ~I and 1 x 55.93 u1 steps, the batch is pipetted to and fro 5 x after the addition and finally pipetted to and fro 5 x with a 100 u1 volume, wait for 15 min. 60 ~g of liposomes (variable, between 1 and 6 ~cg/~I) are mixed with 15 ~g of HA fusion peptide (reduction of the amount to 0.1 ~cg possible) and added to the plasmidIPEI
complex by simple pipetting. For this, the batch is pipetted to and fro 10 x with a 100 ~I 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 experiments. The complexes are stable in the cell culture medium M 199 +
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, PEf, liposome 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.
29/11 '01. 15:02 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU f~038 _ 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.
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 fins! volume of the two constituents mentioned is, depending on the batch type: = a) 465 ~I, b) 245.93 ~I, c) 196.4 ~I, d} 150 p1. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs are likewise possible) is introduced and 15 ~g of plasmid (15 ~g/90 ~l or 75 u1) added thereto. The condensation of the DNA is then carried out by addition of the cationic agent, of 44.55 beg of PEI (N/P ratio 20.7, larger amounts and a reduction to 9.79 fxg are likewise possible). For this, 49.5 u! of PEI solution 0.9 mg/ml are diluted with high-purity water (325.5, 106.43, 5fi.9, 25.5 ~I) and added to the batch in substeps. The addition is carried out in 100 ;~I and/or variable (55.9, 75, 106 u1) 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 ~l volume, wait for 15 min. so ug of liposomes (variable, between 1 and 6 ~gipl) are mixed with 15 pg of HA fusion peptide (reduction of the amount to 0.1 ~g 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 u1 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 180-250 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 300-400 nm).
Example 8: Preparation of a Iiposomal vector complex according to the invention in the sequence plasmid, protamine sulfate, ~PEl~and liposome.
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 p1, b) 320 ~I. Firstly, the buffer (tris 29/1.1 'O1 15:03 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOUDREAU ~ 039 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and p.g of plasmid (15 ug/90 ~I) 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 pg/105 ~I) 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 -~I of PEI solution 0.9 mg/ml are then diluted with high-purity water (106.43, 56.9 1u1) and added to the batch in substeps. The addition is carried out in 700 p! and/or 55.9 p1 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 p1 volume, wait for 15 min. 60 pg of liposomes (variable, between 1 and 6 ~g/~I) 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 u1 volume. The mixture is vortexed. Complexes of similar activity are also obtained by simple mixing of ail components indicated. After preparation, a complex of this type has a size of 180-300 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 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. !n this case, larger or smaller volumes can likewise be chosen.
Example 9: Preparation of a liposomai vector complex according to the invention in the sequence plasmid, protamine sulfate, PEI and liposome having a reduced PEI content.
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: = 319 p1. Firstly, the buffer (tris 10 mM, pH
7.8, olher buffers and pHs likewise possible) is introduced and 15 ~,g of plasmid {15 pgI90 ~I) are added. The condensation of the DNA is then carried out by addition of the protamine sulfate (+I- ratio 3.3, larger or smaller amounts are likewise possible). Protamine sulfate is diluted with 29/1.1 ' Ol 1.5 : 04 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOiIDREAI1 l~J
_ '! 7 high-purity water (29.94 pg/105 ~I) and added to the DNA, the batch being pipetted to and fro 10 x. The complex is allowed to stand for t 5 min for maturation. 30 ~! (NIP ratio 12.5) or 18 ~I (NIP ratio 7.5) of PEI solution 0.9 mg/ml are then diluted to 106 ~I with high-purity water (76 or 88 pf) and added to the batch in substeps. The addition is carried out in 106 p1 steps, after the addition pipette the batch to and fro 10 x each, wait for 15 min. 60 pg of liposomes (variable, between 1 and 6 pgl~l) are mixed with 15 ~g of HA fusion peptide (reduction of the amount to 0.1 ~.g 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/PSIPEI complex by simple pipetting. For this, the batch is pipetted to and fro 10 x with a 100 p1 volume after addition. The mixture is vortexed.
Complexes of similar activity are also obtained by simple mixing of all components indicated.
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 togefher the constituents plasmid DNA and PEI. !n 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 Vii. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and 15 ~g of plasmid (15 pg/90 ~~I) are added thereto. The condensation of the DNA is Then carried out by addition of 44.55 pg of PEI (NIP ratio 20.7, larger amounts and a reduction are likewise possible). For this, 49.5 p1 of PEI solution 0.9 mg/ml are diluted with high-purity water (to 155.93 p1) and added to the batch in substeps.
The addition is carried out in 100 ~I and 55.9 ,~l 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 p1 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 ~g/105 ~I) 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. 60 ug of liposomes (variable, between 1 and 6 ~,g/~I) are mixed with 15 ~g of HA fusion peptide (reduction of the amount to 0.1 ~g 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.
29/11 'O1 15:05 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOUDREAU C~j041 For this, the batch is pipetted to and fro i0 x after the addition with a 100 ~I
volume. The mixture is vortexed. Complexes of similar activity are also obtained by simple mixing of all components indicated.
Example 11: Preparation of a iiposomal vector complex according to the invention in the sequence plasmid, PE1, protamine sulfate and liposome having a reduced PEI 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 final volume of the two constituents mentioned is, depending on the batch type: = 196 u1. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible} is introduced and 15 pg of plasmid (15 ug/90 yi) are added. The condensation of the DNA is then carried out by addition of 9.75 fig, 16.2 ~g or 27 ~g 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 ~I
18 ~I or 30 ~l of PEI solution 0.9 mg/ml are diluted with high-purity water (to 106 pI) and added to the batch while pipetting to and fro 10 x. The 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 ~g1105 ~.I) 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 pg of liposomes (variable, between 1 and 6 ~g/~,I) are mixed with 15 ~g of HA fusion peptide (reduction of the amount to O.i ~g 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 ~l volume.
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 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 29/11 'O1 15:08 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOLTDREALT f~j042 i9 final volume of the two constituents mentioned is 245.93 ~l. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and 15 pg of plasmid (15 pgI90 p1) are added thereto. The condensation of the DNA is then carried out by addition of the cationic agent, 44.55 pg of PEl (NIP ratio 20.7, larger amounts and a reduction are likewise possible). For this, 49.5 p1 of PEI solution 0.9 mglml are diluted with 106.43 ~l of high-purity water and added to the batch in subsieps. The addition is carried out in 1 x 100 fd and 1 x 55.93 ~l 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 ~d volume, wait for 15 min. 75, 45 and 30 ~g of liposomes (between 1 and 6 pglpl) are added to the plasmidIPEI complex by simple pipetting. For this, the batch is pipetted to and fro 10 x after the addition with a 100 p1 volume. The content of the liposomes used can moreover be markedly increased to 10 x the amount.
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 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 p1. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is introduced and 15 ~g of plasmid (15 ug/90 ~l) are added thereto. The condensation of the DNA is then carried out by addition of the cationic agent, 44.55 pg PEl (NIP ratio 20.7, larger amounts and a reduction are likewise possible). For this, 49.5 ~1 of PEI solution 0.9 mg/rnf are diluted with 106.43 w1 of high-purity water and added to the batch in substeps. The addition is carried out in 1 x 100 p! and 1 x 55.93 ~I steps, after the addition the batch is pipetted to and fro 5 x and finally pipetted to and fro 5 x with a 100 u1 volume, wait for 15 min. 60 ~g of liposomes (between 1 and 6 pg/~l) with and without coupled RGD targeter (RGD binds to the a"~ 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 p1 volume.
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 FRCS analysi .
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.
29/11 'O1 ---15:06 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU ~ 043 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 defection of the GFP autofluorescence, 5 luciferase assay, and FACS analysis were then measured. Results of the FACS analysis are compiled in the following table.
f Cells + RGD - RGD
HUVEC rima 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 rostate carcinoma cells 1 1 pC3 rostate carcinoma cells 95 21 HeLa cervical carcinoma cells 4 33 LoVo colon carcinoma cells 8 1 A549 lun carcinoma cells 23 11 MCF-7 breast cancer cells ~3 34 ' JEG-3 chorionic carcinoma cells ~4
Claims (27)
1. A liposomal vector complex comprising the following components a) a nucleic acid sequence of any desired length;
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 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.
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 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 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.
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 component b) is a cationic polymer.
6. A liposomal vector complex as claimed in claim 5, in which the cationic polymer is polyethyleneimine (PEI).
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.
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.
is high-branched-chain.
9. A liposomal vector complex as claimed in claim 6, in which the PEI
is low-branched-chain.
is low-branched-chain.
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 component c) consists of phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, anchor lipid and cholesterol.
12. A liposomal vector complex as claimed in claim 11, the anchor lipid being an N-carboxylphosphatidylethanolamine.
13. A liposomal vector complex as claimed in claim 12, the anchor lipid being an N-glutarylphosphatidylethanolamine.
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.
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, 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.
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.
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.
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 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 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 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 (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 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 formation.
(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 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.
27. A process for the preparation of a liposomal vector complex as claimed in one of claims 1-17, the resulting product being aerosolized.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19925143A DE19925143A1 (en) | 1999-06-02 | 1999-06-02 | New liposomal vector complexes and their use for gene therapy |
| DE19925143.6 | 1999-06-02 | ||
| PCT/EP2000/004678 WO2000074646A2 (en) | 1999-06-02 | 2000-05-23 | Novel liposomal vector complexes and their use in gene therapy |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2375854A1 true CA2375854A1 (en) | 2000-12-14 |
Family
ID=7909923
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002375854A Abandoned CA2375854A1 (en) | 1999-06-02 | 2000-05-23 | Novel liposomal vector complexes and their use in gene therapy |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP1187929A2 (en) |
| JP (1) | JP2003501373A (en) |
| AU (1) | AU4758300A (en) |
| CA (1) | CA2375854A1 (en) |
| DE (1) | DE19925143A1 (en) |
| WO (1) | WO2000074646A2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9387152B2 (en) | 2010-06-28 | 2016-07-12 | The General Hospital Corporation | Blood substitutes and uses thereof |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PT103865A (en) * | 2007-10-25 | 2009-05-15 | Univ De Coimbra | NANO-LIPID BASIC CONVEYORS FOR DIRECT DELIVERY OF VIRAL VECTORS AND PROCESS FOR THEIR PRODUCTION |
| CN101270168B (en) * | 2008-05-13 | 2011-06-22 | 中国药科大学 | Hyaluronic acid stem grafting polyethylene imine copolymer, preparing method and application as genophore |
| CN109996809A (en) | 2016-11-14 | 2019-07-09 | 诺华股份有限公司 | Composition relevant to fusogenic protein MINION, method and therapeutical uses |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6051429A (en) * | 1995-06-07 | 2000-04-18 | Life Technologies, Inc. | Peptide-enhanced cationic lipid transfections |
| FR2739292B1 (en) * | 1995-09-28 | 1997-10-31 | Rhone Poulenc Rorer Sa | PHARMACEUTICAL COMPOSITION USEFUL FOR TRANSFECTING NUCLEIC ACIDS AND USES THEREOF |
| EP0784984B1 (en) * | 1996-01-17 | 2003-07-02 | F. Hoffmann-La Roche Ag | Transfection competent molecules |
| WO1998049350A1 (en) * | 1997-04-30 | 1998-11-05 | Regents Of The University Of Minnesota | In vivo use of recombinagenic oligonucleobases to correct genetic lesions in hepatocytes |
| JP2002510706A (en) * | 1998-04-07 | 2002-04-09 | ロシュ ダイアグノスティックス ゲーエムベーハー | Novel compounds for DNA transfection |
| WO2000003683A2 (en) * | 1998-07-20 | 2000-01-27 | Inex Pharmaceuticals Corporation | Liposomal encapsulated nucleic acid-complexes |
-
1999
- 1999-06-02 DE DE19925143A patent/DE19925143A1/en not_active Withdrawn
-
2000
- 2000-05-23 EP EP00929548A patent/EP1187929A2/en not_active Withdrawn
- 2000-05-23 CA CA002375854A patent/CA2375854A1/en not_active Abandoned
- 2000-05-23 AU AU47583/00A patent/AU4758300A/en not_active Abandoned
- 2000-05-23 JP JP2001501183A patent/JP2003501373A/en not_active Withdrawn
- 2000-05-23 WO PCT/EP2000/004678 patent/WO2000074646A2/en not_active Application Discontinuation
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9387152B2 (en) | 2010-06-28 | 2016-07-12 | The General Hospital Corporation | Blood substitutes and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| AU4758300A (en) | 2000-12-28 |
| JP2003501373A (en) | 2003-01-14 |
| WO2000074646A2 (en) | 2000-12-14 |
| WO2000074646A3 (en) | 2001-08-09 |
| DE19925143A1 (en) | 2000-12-07 |
| EP1187929A2 (en) | 2002-03-20 |
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