Complexes containing plasmid DNA, transferrin-polylysine conjugates, and polylysine-conjugated peptides derived from the N-terminal sequence of the influenza virus hemagglutinin subunit HA-2 have been used for the transfer of luciferase or -galactosidase marker genes to K562 cells, HeLa cells, and BNL CL.2 hepatocytes. These DNA complexes mimic the entry of viruses into cells, as they contain functions for (i) the packaging of the nucleic acid with polylysine, (i) the attachment to the cell and receptor-mediated endocytosis with transferrin as a ligand, and (Mi) the release from endosomes by using membrane-disrupting influenza peptides. The presence of these influenza peptide conjugates in the DNA complexes renders the complexes active in membrane disruption in a liposome leakage assay and results in a substantial augmentation of the transferrin-polylysine-mediated gene transfer.
We have developed a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells. We accomplished this by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, as well as the chicken homologue conalbumin, has been covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with double-stranded DNA, singlestranded DNA, and modified RNA molecules independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and a bacterial plasmid DNA containing the gene for Photinus pyralis luciferase are supplied to eukaryotic cells, high-level expression of the luciferase gene occurs, demonstrating transferrin receptor-mediated endocytosis and expression of the imported DNA. We refer to this delivery system as "transferrinfection."All actively metabolizing cells require iron that is taken up by the cells as a transferrin-iron complex by means of receptormediated endocytosis (the transferrin cycle; refs. 1-3). To exploit this ubiquitous and efficient transport mechanism for introducing DNA into cells, conjugates of chicken transferrin (conalbumin) or human transferrin with DNA-binding polycations were synthesized. We used protamine, a small, naturally occurring, arginine-rich DNA-binding protein (4) and synthetic polylysines of different degrees of polymerization (-90, 270, or 450 L-lysine monomers). As target cells for DNA delivery, avian erythroblasts transformed with conditional erbB oncogenes (temperature sensitive v-erbB or cerbB with or without ligand) were selected because these cells display a particularly active transferrin cycle when induced to differentiate (5, 6). By using this system the transferrin-polycation conjugates were shown to function as high-efficiency iron transporters and as carriers to deliver a gene to the living cell. Polylysine-asialoglycoprotein conjugates have been used similarly to target DNA to liver cells, as reported by Wu and Wu (7,8).MATERIALS AND METHODS Synthesis of Transferrin-Polycation Conjugates. Transferrin was coupled to polylysine as described (9) by ligation through disulfide bonds after modification with the bifunctional reagent succinimidyl 3-(2-pyridyldithio)propionate (SPDP; Pharmacia). The transferrin-protamine conjugates were prepared in a similar manner, except that the less reactive protamine (which lacks lysine amino groups) was modified under nonaqueous conditions. 3-(2-Pyridyldithio)propionate-Modified Transferrin 1. A solution of 120 mg (1.5 t&mol) of chicken transferrin (conalbumin, iron free; Sigma) in 3 ml of 100 mM sodium phosphate buffer (pH 7.8) was subjected to gel filtration on a Seph...
We have previously described a gene delivery system based upon the receptor-mediated endocytosis of DNA complexed with transferrin-polycation conjugates. This delivery system has been found to be very effective for both the internalization and the expression of genetic material in cells that have many transferrin receptors. Upon scrutinization of the parameters involved in this method, which we have termed transfeMnfection, we note two important features of the process: the polycation in polycation-transferrin conjugates, as expected, serves to attach the transferrin moiety to the DNA and, in addition, the polycation functions to condense the DNA into a doughnut structure. Electron microscopic analysis of a range of poorly active to highly active transferrinfection samples reveals a strong correlation between DNA condensation and cellular DNA uptake. Furthermore, we demonstrate that the transfection activity of the DNA complex can be increased by addition of free polycation as long as a sufficient quantity of polycation-transferrin conjugates remains in the complex to ensure its binding to the cellular receptor.Transferrinfection, the cellular uptake and expression of DNA complexed with transferrin (Tf)-polycation conjugates, has been shown to be based on Tf-dependent receptormediated endocytosis (1-3), with the polycation polylysine or protamine acting as the DNA-binding moiety. Consistent with DNA delivery as an endocytotic event, we have demonstrated that the presence of excess free Tf interferes with DNA uptake (2) and that up-regulation of the Tf receptor by agents like desferrioxamine (deferoxamine) increases the subsequent gene expression in K-562 cells (3). Virtually 100% of such cells take up and express a transferrinfected reporter gene (unpublished observation).Our initial studies demonstrated that delivery of DNA occurred maximally by using ratios of Tf-polylysine to DNA that resulted in electroneutrality (1). We chose to examine the composition ofDNA complexes thatgave maximal transferrinfection in some detail with the hope of learning more about the DNA delivery event. We find that there are at least two important features ofa delivery-competent DNA complex: (i) The presence of sufficient polycation in the mixture to ensure full condensation of the DNA molecule into a form that is compatible with endocytosis of the conjugate. We find that we can replace a certain portion of Tf-polycation conjugate with free polycation without compromising DNA delivery, and in certain cases, this replacement can result in enhanced DNA delivery. (ii) The presence of sufficient Tf on the condensed DNA molecule. We must maintain 10-20 Tf molecules per DNA molecule to maintain receptor-mediated gene delivery. When the amount ofTf-polylysine replaced by free polylysine is further increased, DNA delivery falls drastically to low levels typically seen for pure polycation delivery schemes (4). MATERIALS AND METHODSMaterials. Polymers of L-lysine with an average chain length of 55, 90, 200, or 450 lysine residues (Ly...
When a DNA molecule, enzymatically labelled with 32p at one end, is partially digested with a restriction enzyme labelled tdna fragments are obtained which form an overlapping series of molecules, all with a common labelled terminus. ta restriction map can then be constructed from an analysis of the size distribution of these molecules. This technique has been used for the restriction site mapping of cloned histone DNA (h22) where as many as 35 cleavage sites may be accurately determined in a single experiment.
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