Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells.

Abstract

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 Virtually 100% of such cells take up and express a transferrinfected reporter gene (unpublished observation).Our initial studies demonstrated that delivery of DNA occurred maximally by using ratios of Tf-polylysine to DNA that resulted in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...

Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells

“…Complexes in which poly K was substituted with either 3.5% or 7.8% sulfo-LC SPDP gave efficient transfection. These results are consistent with reports that poly K substituted to a high extent with transferrin moieties condensed DNA into complexes which achieve less gene transduction than less substituted poly K. 6 In contrast, Erbacher and colleagues 16 found that polymers 190 residues in length, substituted at 30-40% with galactose moieties, gave maximal expression intensity in the presence of chloroquine. However, the galactose-substituted polymer differs from our system.…”

“…[1][2][3][4][5][6][7][8][9] A carrier, usually composed of a polycationic protein chemically conjugated to a receptor-specific ligand, is used to condense DNA and present it to the target cell. Careful manipulation of the concentration of NaCl can produce complexes, containing a single plasmid DNA molecule, ranging in size from 10-25 nm in diameter.…”

“…However, for receptor-mediated gene transfer, little has been done to examine experimentally the relation of the structure of the protein portion of these complexes to the intensity and duration of gene expression, in part because it is difficult to verify the composition of chemically coupled protein conjugates. Wagner and colleagues 6 studied the relation between the extent of substitution of poly-l-lysine (poly K) with transferrin molecules and the efficacy of gene transfer. More substituted poly K complexes achieved less gene transduction.…”

Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool

“…Molecular conjugates of poly-lysine with natural or artificial ligands utilise the DNA-binding and -condensing properties of poly-lysine to mediate interaction with DNA (Wagner et al, 1991;Perales et al, 1994). Upon formation of a DNA-poly-lysine-ligand complex (polyplex (Felgner et al, 1997)), gene transfer is facilitated via receptor-mediated endocytosis.…”

Gene therapy in the CNS

“…To avoid degradation by the endocytic pathway and to facilitate fusion with the cell membrane, fusion proteins derived from Sendai virus have been incorporated into liposomes. 110,111 To facilitate cytoplasmic transit to the cell nucleus, high mobility group proteins with nuclear localization signals have been complexed to the DNA. 112,113 Most recently, elements from Epstein-Barr virus (oriP and EBNA-1) have been included in the DNA within liposomes to prolong the retention of transgenes in dividing cells.…”