Patent EA-003130-B1 (EA)
Title: Cationic virosomes as transfer system for genetic material
Inventor: WALTI ERNST RUDOLF
IPC: A61K48/00
Filed: 1997-05-04
Abstract: 1. A process for the manufacture of a vesicle having a positively charged lipid bilayer membrane that contains cationic and/or polycationic lipids together with phosphatidylethanolamine or phosphatidylcholine, and at least one functionally active influenza viral hemagglutinin integrated in or covalently linked to the membrane, which is capable of inducing and/or promoting a fusion reaction between the vesicle membrane and a lipid membrane of a target cell, comprising the steps of a) dissolving purified influenza virus with a non-ionic detergent solution and obtaining fraction containing hemagglutinin trimer; b) preparing conjugate molecule complexes consisting of phosphatidylethanolamine (PE), a bifunctional crosslinker, and a cell-specific marker wherein the bifunctional crosslinker on one hand binds to the amino group of PE and on the other hand to a thiol group of the marker, and removing unconjugated material; c) preparing a solution containing: a buffer, a non-ionic detergent; hemagglutinin of influenza virus prepared on pointa), cationic and/or polycationic lipids, phosphatidylethanolamine (PE), and optionally phosphatidylcholine, crosslinker, and at least one cell-specific marker consisting of preformed PE/crosslinker/marker molecule complexes, prepared on stage b), and adjusting the lipid concentrations to - based on total lipids - 90 to 95 % by weight of cationic and/or polycationic lipids and 5 to 10 % by weight of phosphatidylethanolamine; or 45 to 90 % by weight of cationic and/or polycationic lipids, 5 to 10 % by weight of phosphatidylethanolamine and 5 to 50 % by weight of phosphstidylcholine; d) removing the detergent by treating the solution with microcarrier beads, preferably polystyrene Biobeads SM-2, resulting in the formation of positively charged lipid bilayer vesicles; and e) adding to the vesicles a quantity of a desired material for delivery to target cells, sonicating the mixture to integrate the material into the vesicles, and removing non-integrated material, preferably by gel filtration. 2. A process according to claim 1, wherein the weight ratio of hemagglutinin to total membrane lipids is approximately 1 mg/mg or less. 3. A process according to claim 1 or 2, wherein cell-specific marker capable of selective detection and binding of target cells, selected from the group consisting of an antibody, preferably monoclonal antibody, an antibody fragment preferably F(ab')2 fragment or a Fab' fragment, a cytokine and a growth factor. 4. The process according to any of claims 1 to 3, wherein cell-specific marker is Fab' fragment, and the weight ratio of hemagglutinin and said marker is approximately 2:1. 5. The process according to any of claims 1 to 4, wherein the cationic lipids comprise at least one member selected from the group consistins of N-[1,2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N-[1,2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammoniummethylsulfate (DOTAP); N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine; and the polycationic lipids comprise at least one member selected from the group consisting of 1,3-dipalmitoyl-2-phosphatidylethanolamidospermine (DPPES): dioctadecylamidoglycylspermine (DOGS); 2,3-dioleoyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dirnethyl-1 -propaneaminiumtrifluoroacetate (DOSPA); 1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER); and N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butanediammoniumiodide (THOOB). 6. The process according to one or more of claims 1 to 5, wherein said non-ionic detergent is comprise C12E8 or n-oktiloligooxiethylene at a concentration of 10 to 250 mumol per ml of HEPES buffer. 7. The process according to one or more of claims 1 to 6, wherein said microcarrier beads have a wet mesh size of 20 - 50 (0.84 - 0.30 mm) and the solution is treated up to four times with said microcarrier beads. 8. The process according to one or more of claims 1 to 7, wherein said crosslinking agent is a heterobifunctional organic molecule, preferably having at least one maleimido and at least one carboxyl group and being selected from the group consisting of bis-N-succinimidyl derivatives and photoactivatable succinimidyl derivatives. 9. The process according to claim 1-8, wherein incompletely conjugated material, particularly unreacted complexes of PE/crosslinking agent N-[4-(p-maleimidophenyl)-butyryll-phosphatidyl-ethanolamine (MP8.PE) and 4-(N-maleimidomethyl)cyclohexane-1-carboxylate-phosphatidylethanolamine (MCC.PE), are separated from the reaction products by gel chromatography, preferably by affinity chromatography with an agarose matrix, most preferably by reduced Thiopropylsepharose 6B. 10. The process according to any of claims 1 to 9, wherein said desired material for delivery is genetic material selected from the group consisting of short chain DNA or RNA, deoxyribonucleotides, oligodeoxyribonucleotides, oligodeoxyribonucleotide selenoates, oligodeoxyribonucleotide phosphorothioates, oligodeoxyribonucleotide phosphoramidates, oligodeoxyribonucleotide methylphosphonates, peptide nucleic acids, ribonucleotides, oligoribonucleotides, oligoribonucleotide phosphorothioates, 2'-OMe-oligoribonucleotide phosphates, 2'-OMe-oligoribonuclsotide phosphorothioates, ribozymes, genes, plasmids and vectors. 11. A lipid vesicle having positively charged lipid bilayer membrane which comprises cationic and/or polycationic lipids together with, at least one functionally active viral influenza hemagglutinin integrated in or covalently linked to the membrane, which is capable of inducing and/or promoting a fusion reaction between the vesicle membrane and a lipid membrane of a target cell, comprising the steps of a) vesicle membrane contains - 90 to 95 % by weight of cationic and/or polycationic lipids and 5 to 10 % by weight of phosphatidylethanolamine; or 45 to 90 % by weight of cationic and/or polycationic lipids, 5 to 50 % by weight of phosphstidylcholine, and 5 to 10 % by weight of phosphatidylethanolamine; b) membrane contains, at least one bifunctional crosslinking agent, linked with membrane by covalent bonding with phosphatidylethanolamine; c) vesicle contains, at least one cell-specific marker capable of selective detection and binding of target cells, selected from the group consisting of an antibody, preferably monoclonal antibody, an antibody fragment preferably F(ab')2 fragment or a Fab' fragment, a cytokine and a growth factor, wherein said marker is linked with membrane using said crosslinking agent and crosslinking agent reacts with the vacant carbothiolic cell-specific group; and d) Vesicle has necessary genetic material for delivery to target cells. 12. Vesicle according to claims 11, wherein the cationic lipids comprise at least one member selected from the group consistins of N-[1,2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N-[1,2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammoniummethylsulfate (DOTAP); N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine; and the polycationic lipids comprise at least one member selected from the group consisting of 1,3-dipalmitoyl-2-phosphatidylethanolamidospermine (DPPES): dioctadecylamidoglycylspermine (DOGS); 2,3-dioleoyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dirnethyl-1 -propaneaminiumtrifluoroacetate (DOSPA); 1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER); and N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butanediammoniumiodide (THOOB). 13. Vesicle according to any of claims 11or 12, wherein membrane comprise 1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER) as a polycationic lipid. 14. Vesicle according to any of claims 11or 13, wherein the genetic material entrapped in said vesicle is selected from the group consisting of short chain DNA or RNA, deoxyribonucleotides, oligodeoxyribonucleotides, oligodeoxyribonucleotide selenoates, oligodeoxyribonucleotide phosphorothioates, oligodeoxyribonucleotide phosphoramidates, oligodeoxyribonucleotide methylphosphonates, peptide nucleic acids, ribonucleotides, oligoribonucleotides, oligoribonucleotide phosphorothioates, 2'-OMe-oligoribonucleotide phosphates, 2'-OMe-oligoribonucleotide phosphorothioates, ribozymes, genes, plasmids and vectors. 15. The vesicle according to claims 11 to 14, wherein its diameter is in the range of 120 - 180 nm. 16. Vesicle according to any of claims 11or 15 is prepared according to any of 1 to 10. 17. Lipid vesicle as defined in any of claims 11 to 16, is applied as a carrier system for drug delivery, preferably for specific and non-infections delivery of desired genetic material to target cells or tissues, particularly to resting or proliferating mammalian cells. 18. Application according to claim 17 wherein the delivery of genetic material is preferably for the prophylactic and/or therapeutic treatment of humans or animals. 19. Application according to any of claims 17 or 18, wherein genetic material preferably comprises at least one antisense oligonucleotide suitable for antisense therapy, for the treatment of a disease selected from the group consisting of cancer, leukemia, and viral infection. 20. Application according to claim 19, wherein the antisense oligonucleotide is targeting protooncogene or oncogene encoded mRNA. 21. A method of manufacturing of molecule complex consisting of phosphatidylethanolamine (PE), heterobifunctional cell-specific crosslinker, selected from the group consisting of an antibody, an antibody fragment, a cytokine and a growth factor, comprising the following steps: a) dissolving of cell-specific marker, preferably Fab' fragment of monoclonal antibody, in buffer solution, preferably comprising 100 mM NaCl, 40 mM of citric acid, 35 mM Na2HPO4 x 2H2O, 2 mM of EDTA and pH 5.5; b) mixing of obtained solution with detergent solution, which comprises 0.5% of non-ionic detergent, preferably n-octyl-olisooxyethylene in citrate buffer, and with prepared in advance structures, consisting o