A multi-domain protein for beta(1) integrin-targeted DNA delivery

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)(4)inv, that is designed to target plasmid DNA t o beta(1) integrins in remodeling tissue. If contains a nonspecific DNA-bin ding domain (SPKR)(4), a rigid or-helical linker, and the C-terminal beta(1 ) integrin binding domain (aa 793-987) of the Yersinia pseudotuberculosis i nvasin protein. (SPKR)(4)inv could be purified at high yields using a bacte rial;expression system. We show that (SPKR),inv binds with high affinity to both plasmid DNA and beta(1) integrins. in a cell attachment assay, the ap parent affinity of (SPKR)(4)inv for beta(1) integrins is three orders of ma gnitude higher than that of the synthetic peptide integrin ligand RGDS. (SP KR)(4)inv-plasmid complexes are not active in an in vitro transfection assa y. However, transfection efficiencies of plasmid complexes with a cationic lipid micelle (DOTAP/Tween-20) or a cationic polymer (polyethylenimine), ar e significantly increased in combination with (SPKR),inv. (SPKR),inv-mediat ed transfection can be inhibited by a soluble form of beta(1) integrin, whi ch is evidence for ifs receptor specificity. In conclusion, (SPKR),inv allo ws PI integrin-specific targeting of plasmid-carrier complexes, while avoid ing inefficient and cumbersome coupling chemistry. The modular design of th e 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.