| Autor: |
Fortunati, E, Ehlert, E, van Loo, N-D, Wyman, C, Eble, J A, Grosveld, F, Scholte, B J |
| Předmět: |
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| Zdroj: |
Gene Therapy; Sep2000, Vol. 7 Issue 17, p1505, 11p |
| Abstrakt: |
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)[sub 4]inv, that is designed to target plasmid DNA to β[sub 1] integrins in remodeling tissue. It contains a nonspecific DNA-binding domain (SPKR)[sub 4], a rigid α-helical linker, and the C-terminal β[sub 1] integrin binding domain (aa 793-987) of the Yersinia pseudotuberculosis invasin protein. (SPKR)[sub 4]inv could be purified at high yields using a bacterial expression system. We show that (SPKR)[sub 4]inv binds with high affinity to both plasmid DNA and β[sub 1] integrins. In a cell attachment assay, the apparent affinity of (SPKR)[sub 4]inv for β[sub 1] integrins is three orders of magnitude higher than that of the synthetic peptide integrin ligand RGDS. (SPKR)[sub 4]inv-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)[sub 4]inv. (SPKR)[sub 4]inv-mediated transfection can be inhibited by a soluble form of β[sub 1] integrin, which is evidence for its receptor specificity. In conclusion, (SPKR)[sub 4]inv allows β[sub 1] 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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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