Article

A multi-domain protein for beta1 integrin-targeted DNA delivery.

Department of Cell Biology, Erasmus University, Rotterdam, The Netherlands.
Gene Therapy (Impact Factor: 4.2). 10/2000; 7(17):1505-15. DOI: 10.1038/sj.gt.3301258
Source: PubMed

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 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.

0 Followers
 · 
76 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Lack of safe, efficient and controllable methods for delivering therapeutic genes appears to be the most important factor preventing human gene therapy. Safety issues encountered with viral vectors have prompted substantial attention to in vivo investigations with non-viral vectors throughout the past decade. However, developing non-viral vectors with effectiveness comparable to viral ones has been a challenge. The strategy of designing multifunctional synthetic carriers targeting several extra- and intracellular barriers in the gene transfer pathway has emerged as a promising approach to improving the efficacy of gene delivery systems. This review will explain how sophisticated synthetic vectors can be created by combining conventional polycationic vectors such as polyethylenimine and basic amino acid peptides with additional polymers and peptides that are designed to overcome potential barriers to the gene delivery process.
    International Journal of Pharmaceutics 09/2013; 457(1). DOI:10.1016/j.ijpharm.2013.09.014 · 3.79 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Successful gene delivery into mammalian cells using bactofection requires entry of the bacterial vector via cell surface integrin receptors followed by release of plasmid DNA into the cellular environment. We show, for the first time, that addition of the DNA transfection reagent Lipofectamine improves entry of invasive Escherichia coli into HeLa cells and enhances up to 2.8-fold green fluorescent protein (GFP) expression from a reporter plasmid. The addition of Lipofectamine may be applicable to other bacterial vectors to increase their DNA delivery efficiency into mammalian cells.
    Analytical Biochemistry 08/2013; 439(2):142–144. DOI:10.1016/j.ab.2013.04.010 · 2.31 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Gene delivery and therapy in general The concept of gene therapy is a promising approach towards clinical treatment of pathological processes. The underlying principle of gene therapy is based on the introduction of genetic material into living cells in order to achieve a therapeutic biological effect [1]. Generally, this involves introducing DNA encoding a gene for a therapeutic protein. In somatic gene therapy the target cells are not part of the germ line and therefore the effects are restricted to the individual. In contrast, in germ line gene therapy the egg or sperm cells are manipulated and thus the potential future offspring of the individual are effected. For this reason, germ line gene therapy is not permitted under current legislation [2]. Gene therapy offers the potential of correcting the underlying cause of hereditary monogenetic diseases such as cystic fibrosis (CF) [3] and haemophilia B [4], for which the responsible gene is known. Therapeutic benefits of gene therapy can be expanded to a wide range of diseases that are not strictly hereditary, such as cancer [5] and cardiovascular diseases [6]. In addition, applications of gene therapy can reach much further: introducing disease-modifying genes into already dysfunctional organs may alter the course of disease [7]. When an infectious agent is involved, gene therapy can be directed towards elimination of the agent from the organism or towards prevention of infection in the form of vaccination [8]. Recent developments indicate that besides delivery of dsDNA, other applications such as delivery of RNA sequences, RNAi, and RNA decoys that bind regulatory proteins [9] offers potential. For example, infectious agents such as HIV [10] and Hepatitis C virus [11] are targets for the development of RNAi for virus inhibition.

Full-text (2 Sources)

Download
9 Downloads
Available from
May 15, 2014