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

被引:55
作者
Fominaya, J [1 ]
Uherek, C [1 ]
Wels, W [1 ]
机构
[1] Inst Expt Canc Res, Tumor Biol Ctr, D-79106 Freiburg, Germany
关键词
gene therapy; non-viral gene delivery; fusion protein; TGF-alpha; EGF receptor; Pseudomonas exotoxin A; GAL4;
D O I
10.1038/sj.gt.3300614
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Fusion proteins engineered to incorporate distinct functions which co-operate in mediating the cell-type specific uptake and intracellular delivery of DNA present an attractive approach for the development of self-assembling vector systems for targeted gene transfer. Here we have chosen the EGF receptor overexpressed in many human tumors of epithelial origin as a target for a novel modular fusion protein. We have fused a cDNA fragment of the human EGF receptor ligand TGF-alpha to sequences encoding the translocation domain of Pseudomonas exotoxin A as an endosome escape activity, and the DNA-binding domain of the yeast GAL4 transcription factor Upon bacterial expression, this TEG fusion protein displayed specific binding to EGF receptors. Complexes of the chimeric protein and plasmid DNA carrying a luciferase reporter gene, after condensation with poly-L-lysine resulted in an up to 150-fold increase in reporter gene expression in EGF receptor expressing cells in comparison to poly-L-lysine-DNA complexes alone. While in COS-1 cells no additional endosome escape activity was required, in A431 cells gene delivery was dependent on the simultaneous presence of the endosome destabilizing reagent chloroquine indicating that cell-type specific factors such as different intracellular routing of protein-DNA complexes greatly influence transfection efficiency.
引用
收藏
页码:521 / 530
页数:10
相关论文
共 33 条
[31]   EGF RECEPTOR AND P185(ERB-2)-SPECIFIC SINGLE-CHAIN ANTIBODY TOXINS DIFFER IN THEIR CELL-KILLING ACTIVITY ON TUMOR-CELLS EXPRESSING BOTH RECEPTOR PROTEINS [J].
WELS, W ;
BEERLI, R ;
HELLMANN, P ;
SCHMIDT, M ;
MARTE, BM ;
KORNILOVA, ES ;
HEKELE, A ;
MENDELSOHN, J ;
GRONER, B ;
HYNES, NE .
INTERNATIONAL JOURNAL OF CANCER, 1995, 60 (01) :137-144
[32]  
WELS W, 1998, SELF ASSEMBLING COMP, P351
[33]   RECEPTOR-MEDIATED ENDOCYTOSIS OF TRANSFERRIN POLYCATION CONJUGATES - AN EFFICIENT WAY TO INTRODUCE DNA INTO HEMATOPOIETIC-CELLS [J].
ZENKE, M ;
STEINLEIN, P ;
WAGNER, E ;
COTTEN, M ;
BEUG, H ;
BIRNSTIEL, ML .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (10) :3655-3659