A multi-domain protein for beta1 integrin-targeted DNA delivery.
Gene Therapy , Volume 7 - Issue 17 p. 1505- 1515
The development of effective receptor-targeted nonviral vectors for use in vivo is complicated by a number of technical problems. One of these is the low efficiency of the conjugation procedures used to couple protein ligands to the DNA condensing carrier molecules. We have made and characterized a multi-domain protein (SPKR)4inv, that is designed to target plasmid DNA to beta1 integrins in remodeling tissue. It contains a nonspecific DNA-binding domain (SPKR)4, a rigid alpha-helical linker, and the C-terminal beta1 integrin binding domain (aa 793-987) of the Yersinia pseudotuberculosis invasin protein. (SPKR)4inv could be purified at high yields using a bacterial expression system. We show that (SPKR)4inv binds with high affinity to both plasmid DNA and beta1 integrins. In a cell attachment assay, the apparent affinity of (SPKR)4inv for beta1 integrins is three orders of magnitude higher than that of the synthetic peptide integrin ligand RGDS. (SPKR)4inv-plasmid complexes are not active in an in vitro transfection assay. However, transfection efficiencies of plasmid complexes with a cationic lipid micelle (DOTAP/Tween-20) or a cationic polymer (polyethylenimine), are significantly increased in combination with (SPKR)4inv. (SPKR)4inv-mediated transfection can be inhibited by a soluble form of beta1 integrin, which is evidence for its receptor specificity. In conclusion, (SPKR)4inv allows beta1 integrin-specific targeting of plasmid-carrier complexes, while avoiding inefficient and cumbersome coupling chemistry. The modular design of the expression vector allows production of similar multi-domain proteins with a different affinity. The further development of such complexes for use in vivo is discussed.
|Antigen-Antibody Reactions, Antigens, CD29/*metabolism, Fatty Acids, Monounsaturated, Gene Therapy/*methods, Genetic Engineering, Genetic Vectors/*metabolism, Humans, Plasmids/metabolism, Polyethyleneimine, Protein Binding, Quaternary Ammonium Compounds, Transfection/*methods, Tumor Cells, Cultured, Yersinia pseudotuberculosis/*genetics|
|Organisation||Erasmus MC: University Medical Center Rotterdam|
Fortunati, E, Ehlert, E.M.E, van Loo, N.D, Wyman, C, Eble, J.A, Grosveld, F.G, & Scholte, B.J. (2000). A multi-domain protein for beta1 integrin-targeted DNA delivery. Gene Therapy, 7(17), 1505–1515. doi:10.1038/sj.gt.3301258