A multi-domain protein for β1 integrin-tangeted DNA delivery

Department of Cell Biology, Erasmus University, Rotterdam, The Netherlands.
Gene Therapy (Impact Factor: 3.1). 10/2000; 7(17):1505-15. DOI: 10.1038/sj.gt.3301258
Source: PubMed


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.

Download full-text


Available from: Bob J Scholte, Mar 13, 2014
  • Source
    • "This has important consequences for the engineering of tissue supports that, to date, generally involves RGD conjugation to polymers in a random distribution or non-specific adsorption of collagen or fibronectin (Rowley et al 1999; Tan et al 2001; Yang et al 2001). Similarly, recombinant proteins and polypeptides binding integrin for targeted gene delivery via receptor-mediated endocytosis have focussed on monovalent RGD and invasin ligands, extended by cationic polypeptides binding DNA (Harbottle et al 1998; Fortunati et al 2000; Kunath et al 2003). As our understanding of integrin–ligand binding advances, it would appear timely to explore alternative polypeptide templates facilitating a clustered organisation of RGD ligands. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Monovalent RGD (arginine-glycine-aspartic acid) peptides or polymers furnished with RGD in random distributions are employed as cell-scaffolds and gene delivery vehicles. However, integrin binding to RGD is dependent on the spatial distribution (clustering) of the ligand and intrinsic integrin affinity via conformational changes (avidity). Here we have designed and expressed a polypeptide consisting of a tetrameric coiled coil and spacer facilitating polyvalent (clustered) display of integrin ligands; the RGD motif was used as proof of principle. Size-exclusion chromatography and circular dichroism showed that the polypeptide self assembled as a tetramer in solution with a defined secondary structure. Cell adhesion to surfaces coated with the polypeptide was up to 3-fold greater than that for (monovalent) RGDS peptide at equivalent concentrations. Moreover, the polypeptide in solution at concentrations >or= 1 microM inhibited cell adhesion to fibronectin-coated surfaces, while RGDS peptide in solution at concentrations up to 500 muM did not. These cell data demonstrate that the polypeptide bound integrin receptors in a polyvalent manner. The polypeptide will therefore be of use in the engineering of tissue-culture scaffolds with increased cell adhesion activity, or to targeted gene delivery vehicles, and could incorporate protein ligands in place of the RGD motif.
    Journal of Pharmacy and Pharmacology 08/2006; 58(7):959-66. DOI:10.1211/jpp.58.7.0011 · 2.26 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The adenoviral vector is one of the most used viral vectors for gene therapy applications. The specificity towards its primary receptor is a limitation of the use of HAdV as therapeutical vector, as not all cells have these primary receptors on their surface. To overcome this problem scientists have started to change the receptor specificity of the HAdV vectors by introducing heterologous polypeptides. One of the most promising candidates to function as anchor for these heterologous polypeptides is the minor capsid protein IX (pIX). Alpha-helical spacers of different lengths have been inserted between pIX and a polypeptide ligand. The results showed that an increase in spacer length positively correlated with the accessibility of the peptide on the capsid surface for antibodies and cellular membrane proteins. Next, a hyper stable scFv directed against -galactosidase was incorporated in the virion via fusion to pIX. These data were the first to show incorporation of a hyper-stable scFv into the HAdV, and that was functional on the capsid surface. Beside the technical approach to use pIX as anchor for heterologous polypeptides here also the function of pIX in the capsid has been studied. This study has led to the model in which the capsid stability mediated by pIX might not be the result of homo-multimerization of this molecule.
  • [Show abstract] [Hide abstract]
    ABSTRACT: We have isolated several organ- and tumor-homing peptides by using in vivo phage display. This technology involves the screening of peptide libraries in a living animal. The peptides that result from such a selection home to specific organs or tissues because they recognize molecular ‘addresses’, receptors that are differentially expressed in vascular beds. Targeted delivery of chemotherapeutics, pro-apoptotic peptides and cytokines to tumors using these peptides improved therapeutic efficacy in animal models. Translation of this technology into clinical applications will form the basis for targeting therapeutic and imaging agents in the context of cancer and other diseases.
    Current Opinion in Chemical Biology 06/2001; 5(3-5):308-313. DOI:10.1016/S1367-5931(00)00207-6 · 6.81 Impact Factor
Show more