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Abstract
Potential problems with the use of viral vectors for gene therapy necessitate the development of efficient nonviral vectors. The association of transferrin, or the pH-sensitive peptide GALA, with cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane and its equimolar mixture with dioleoylphosphatidylethanolamine, under conditions where the liposome/DNA complex is negatively charged, drastically increased luciferase expression from pCMVluc. The percentage of cells transfected, measured by beta-galactosidase expression, was also increased by about 10-fold. The zeta potential of the ternary complexes was lower than that of the liposome/DNA complexes. Transfection activity of positively charged complexes was also enhanced by association with transferrin, GALA or the influenza hemagglutinin N terminal peptide HA-2, but to a smaller extent compared with the negatively charged complexes. The enhancement of gene delivery by transferrin or GALA was not affected significantly by the presence of serum and did not cause significant cytotoxicity. Our results indicate that negatively charged ternary complexes of cationic liposomes, DNA and transferrin, or fusigenic peptides, can facilitate efficient transfection of cultured cells, and that they may alleviate the drawbacks of the use of highly positively charged complexes for gene delivery in vivo.
Highlights
The feasibility of gene therapy for the treatment of genetic metabolic disorders, cancer and AIDS has been demonstrated in recent studies
Our results indicate that net negatively charged ternary complexes of transferrin/cationic liposomes/DNA can be more effective in mediating gene delivery than positively charged complexes
We investigated the effect on transfection of two different synthetic fusigenic peptides, ‘GALA’33,34 and the influresults have been presented previously in preliminary form.[38,39] lipid/DNA (+/−) charge ratios were tested, luciferase
Summary
The feasibility of gene therapy for the treatment of genetic metabolic disorders, cancer and AIDS has been demonstrated in recent studies. Cationic liposomes, have been used extensively for in vitro and in vivo gene delivery, and constitute a viable alternative to viral gene delivery vehicles.[14,16,17] Using this delivery system, relatively stable expression has been achieved in a number of tissues.[18,19,20,21,22,23] liposomal vectors have several advantages, including lack of immunogenicity, safety, ability to package large DNA molecules and ease of preparation,[14,15,17] they have a limited efficiency of delivery and gene expression, toxicity at higher concentrations, potentially adverse interactions with biological milieux rich in negatively charged macromolecules, and inability to reach tissues beyond the vasculature unless directly injected into the tissue.[15,24,25,26,27] Genes delivered in vivo via cationic liposomes are expressed primarily in cells of the vascular compartment,[20,21] transgene expression has been noted in deep tissues.[20,22] Cationic liposome–DNA complexes (‘lipoplexes’28) may be coated with serum proteins such as lipoproteins and albumin, or bind nonspecifically to cells such as erythrocytes, lymphocytes and endothelial cells, as well as to extracellular matrix proteins This may limit the ability of the complexes to reach target tissues and cells.[14,16,24]. Our results indicate that net negatively charged ternary complexes of transferrin/cationic liposomes/DNA can be more effective in mediating gene delivery than positively charged complexes
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