Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary cells in the presence of adenovirus

Endotoxin (lipopolysaccharide, LPS) is commonly found as a contaminant in plasmid DNA preparations. We demonstrate here that the quantities of LPS typically contaminating DNA preparations can generate a toxicity to primary cells (primary human skin fibroblasts, primary human melanoma cells) in the presence of entry-competent adenovirus particles. Toxicity can be observed with as little as 100 ng/ml free LPS or 100 pg/ml LPS when the LPS is assembled into polylysine/adenovirus complexes. Simple and effective methods of removing the contaminating LPS using either a polymyxin B resin or Triton X-114 extraction are described. Treatment of DNA samples to remove LPS eliminates the toxicity to primary cells

DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules for the introduction of genes into eucariotic cells. We describe here a more specific method for conjugation of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNAbinding domains. Successful transferrin-receptor-mediatedd elivery and expression of the Photinus pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation method facilitates the synthesis of large quantities (>lo0 mg) of conjugates. INTRODUCTION Transferrin-polycation conjugates are efficient carriers for the uptake of DNA into eucariotic cells (I). This gene transfer technique, termed tramferrinfection, is based on receptor-mediated endocytosis of DNA complexed with polycation-transferrin conjugates (2,3). Our initial conjugate synthesis (1) involved the modification of one to two amino groups on the transferrin molecule with the bifunctional reagent succinimidyl34 2-pyridy1dithio)propionate (SPDP), followed by ligation to similarly modified polycations (polylysine or protamine) through the formation of disulfide bonds. Because there are more than 50 lysines on the large (about 80 kDa) transferrin protein, the actual site (or sites) of ligation to the polycation is unknown with this method. In this paper we describe the synthesis of new transferrin conjugates that are ligated with DNA-binding compounds in a specific manner through modification of the transferrin carbohydrate moiety. The conjugates thus obtained are free of any groups derived from chemical linking agents, since the connecting atoms are already present within the starting compounds. The carbohydrate group acts as anatural spacer that puts a 32-atom distance between the transferrin and the DNA binding moiety. This spacer effect may be important for appropriate presentation of the ligand to its receptor. As a DNA-binding compound, the polycation polylysine was used, similar to the use described in ref 1 or to the asialo-orosomucoid conjugates prepared by Wu and Wu (4). We have also prepared a novel type of transferrin conjugate that contains the DNA intercalator ethidium homodimer (5) as the DNAbinding group and demonstrate successful receptormediated gene delivery with these conjugates. EXPERIMENTAL PROCEDURES Human transferrin (iron-free), conalbumin (iron-free), and poly(L-lysine) were obtained from Sigma. Liquid chro- Abbreviations used: FITC, fluorescein ieothiocyenate; TfEtD, traneferrin-ethidium homodimer conjugate; TfpL, traneferrinpolytL- lysine) conjugate; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid

Transferrin-polycation conjugates as carriers for DNA uptake into cells.

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, single-stranded 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.

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

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 transferrinfection, 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

High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles.

The Boltzmann transport equation is commonly considered to be the best semi-classical description of carrier transport in semiconductors, providing precise information about the distribution of carriers with respect to time (one dimension), location (three dimensions), and momentum (three dimensions). However, numerical solutions for the seven-dimensional carrier distribution functions are very demanding. The most common solution approach is the stochastic Monte Carlo method, because the gigabytes of memory requirements of deterministic direct solution approaches has not been available until recently. As a remedy, the higher accuracy provided by solutions of the Boltzmann transport equation is often exchanged for lower computational expense by using simpler models based on macroscopic quantities such as carrier density and mean carrier velocity. Recent developments for the deterministic spherical harmonics expansion method have reduced the computational cost for solving the Boltzmann transport equation, enabling the computation of carrier distribution functions even for spatially three-dimensional device simulations within minutes to hours. We summarize recent progress for the spherical harmonics expansion method and show that small currents, reasonable execution times, and rare events such as low-frequency noise, which are all hard or even impossible to simulate with the established Monte Carlo method, can be handled in a straight-forward manner. The applicability of the method for important practical applications is demonstrated for noise simulation, small-signal analysis, hot-carrier degradation, and avalanche breakdown

Transferrinfection. A Highly Efficient Way to Express Gene Constructs in Eukaryotic Cells

No abstract available

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAcα1-O-Ser/Thr, where GalNAc is N-acetyl-D-galactosamine) is expressed in many carcinomas, in haemopoietic disorders including the Tn syndrome, and on human immunodeficiency virus (HIV) coat glycoproteins, but is not expressed on normal, differentiated cells because of the expression of a Tn-processing galactosyltransferase. Using Jurkat T leukaemic cells which express high levels of Tn antigen due to deficient Tn galactosylation, we have established the Tn antigen-mediated gene transfer and demonstrate the considerable efficiency of this approach. We used poly(L-lysine) conjugates of the monoclonal antibody 1E3 directed against the Tn antigen to deliver the luciferase and β-galactosidase reporter genes to Jurkat cells by receptor-mediated endocytosis. Addition of unconjugated 1E3 reduced transfection efficiency in a concentration-dependent manner and incubation with free GalNAc abolished DNA transfer completely, indicating that gene delivery is indeed mediated by the Tn antigen. Pre-treatment of Jurkat cells with Vibrio cholerae sialidase, which uncovers additional Tn antigens, resulted in an improvement of gene transfection. Both human and chicken adenovirus particles attached to the DNA/polylysine complex strongly augmented transgene expression. When the β-galactosidase (lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis, up to 60% of the cells were positive in the cytochemical stain using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) as a chromogenic substrate. The efficiency of the transferrin receptor-mediated DNA uptake into Jurkat cells was comparatively low, although these cells were shown to express considerable amounts of transferrin receptor. We show here that a mucin-type carbohydrate antigen mediates highly efficient DNA uptake by endocytosis into Jurkat T cells. This method represents a 50-fold improvement of Jurkat cell transfection efficiency over other physical gene transfer techniques. Specific gene delivery to primary cancer cells exhibiting Tn epitopes may especially be desirable in immunotherapy protocol