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  • Nonviral Transfer Technology
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A multi-domain protein for β1 integrin-targeted DNA delivery

Gene Therapy volume 7, pages 1505–1515 (2000)Cite this article

Abstract

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 β1 integrins in remodeling tissue. It contains a nonspecific DNA-binding domain (SPKR)4, a rigid α-helical linker, and the C-terminal β1 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 β1 integrins. In a cell attachment assay, the apparent affinity of (SPKR)4inv for β1 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 (polyethyl- enimine), are significantly increased in combination with (SPKR)4inv. (SPKR)4inv-mediated transfection can be inhibited by a soluble form of β1 integrin, which is evidence for its receptor specificity. In conclusion, (SPKR)4inv allows β1 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.

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Acknowledgements

This project was funded in part by the Dutch foundation for technical research STW (project RNG 55.3900) and the Erasmus University. We thank Marcel Sluijter (Lab Pediatrics, SKZ, Rotterdam) for the isolation of Yersinia pseudotuberculosis DNA.

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Author notes

  1. E Fortunati

    Present address: Department Pulmonary Diseases, University Hospital Utrecht, The Netherlands

Authors and Affiliations

  1. Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands

    E Fortunati, E Ehlert, N-D van Loo, C Wyman, F Grosveld & B J Scholte

  2. Institute for Physiological Chemistry, University of Münster, Germany

    J A Eble

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Fortunati, E., Ehlert, E., van Loo, ND. et al. A multi-domain protein for β1 integrin-targeted DNA delivery. Gene Ther 7, 1505–1515 (2000). https://doi.org/10.1038/sj.gt.3301258

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