A chimeric fusion protein containing transforming growth factor-α mediates gene transfer via binding to the EGF receptor

Abstract

Fusion proteins engineered to incorporate distinct functions binding to EGF receptors. Complexes of the chimeric prowhich co-operate in mediating the cell-type specific uptake tein and plasmid DNA carrying a luciferase reporter gene, and intracellular delivery of DNA present an attractive after condensation with poly-L-lysine resulted in an up to approach for the development of self-assembling vector 150-fold increase in reporter gene expression in EGF systems for targeted gene transfer. Here we have chosen receptor expressing cells in comparison to poly-L-lysinethe EGF receptor overexpressed in many human tumors DNA complexes alone. While in COS-1 cells no of epithelial origin as a target for a novel modular fusion additional endosome escape activity was required, in protein. We have fused a cDNA fragment of the human A431 cells gene delivery was dependent on the simul-EGF receptor ligand TGF-␣ to sequences encoding the taneous presence of the endosome destabilizing translocation domain of Pseudomonas exotoxin A as an reagent chloroquine indicating that cell-type specific facendosome escape activity, and the DNA-binding domain of tors such as different intracellular routing of protein-DNA the yeast GAL4 transcription factor. Upon bacterial complexes greatly influence transfection efficiency. expression, this TEG fusion protein displayed specific

Cell delivery, intracellular trafficking and expression of an integrin-mediated gene transfer vector in tracheal epithelial cells

“…6 This sug- , exposure to hypertonic medium and different drugs, all of which are known to modify endocytosis, on internalization and expression of the complex. The uptake of 35 S-labeled pDNA and subsequent luciferase expression of the transgene were investigated (Figure 3a and b, respectively). Uptake was determined by subtracting the binding at 4°C from the cell association (binding and internalization) at 37°C.…”

“…To determine further the subcellular localization of vector we performed subcellular fractionation on Percoll gradients of cells incubated with 35 S-labeled pDNA with or without the [K] 16 RGD peptide and/or lipofectamine. However, we first characterized the gradient of homogenized cells and observed that the upper gradient fractions contained early and late endosomes at a density of about 1.03 and 1.04, respectively, while the lower fractions contained lysosomes at a density of about 1.10.…”

Intracellular trafficking of nonviral vectors

“…Ligand choice is largely dictated by whether or not the target receptor undergoes endocytosis. Among the additional ligands that have been tested are TGFa, 21 insulin, 22,23 folate, [24][25][26] basic FGF, 27 integrin-binding motifs, [28][29][30] and heregulin. 31 Antibodies generated against certain cell-specific receptors also have been used to direct gene transfer.…”

3PO, a novel nonviral gene delivery system using engineered Ad5 penton proteins

“…Directing nonviral vectors to cellular receptors by the incorporation of specific ligands, such as transferrin, asialoglycoprotein, or epithelial growth factor (EGF), has provided an efficient means of crossing the plasma membrane by receptor-mediated internalization. [1][2][3][4][5][6][7][8][9][10][11][12][13] The cellular endosome encapsulating the vector following endocytosis, however, has proven to be a major barrier to nuclear delivery and gene expression by nonviral vectors. 14 Without endosome penetration, lysosomal enzymes eventually degrade the DNA, resulting in low transduction.…”