An RGD–Oligolysine Peptide: A Prototype Construct for Integrin-Mediated Gene Delivery

Abstract

We have synthesized a linear, bifunctional peptide that comprises an integrin-targeting domain containing an arginine–glycine–aspartic acid tripeptide motif and a DNA-binding moiety consisting of a short stretch of 16 lysine residues. This peptide can form distinctive, condensed complexes with DNA and is capable of mediating its delivery and expression in a variety of mammalian cells in culture. Internalization is mediated by cell surface integrin receptors via a mechanism that is known to be phagocytic. We have analyzed the relationship between DNA and peptide and have investigated the conditions suitable for optimal gene delivery. The formation of condensed peptide DNA complexes leads to resistance to nuclease degradation. The level of reporter gene expression obtained is dependent on the peptide-to-DNA ratio and is enhanced in the presence of the endosomal buffer chloroquine, polyethyleneimine, and deactivated adenovirus during gene delivery. Under optimal conditions the levels of reporter gene expression obtained approach or even exceed those obtained with DNA delivered with the commercial liposome Lipofectamine. The ability to produce an efficient gene delivery system using small, easily modified, and well-defined constructs that have no constraint of particle size demonstrates the advantages of integrin-targeting peptides for gene transfer. This article investigates the use of a small synthetic peptide, [K]₁₆RGD, as a receptor-mediated gene delivery vehicle. [K]₁₆RGD is a bifunctional linear peptide that comprises an integrin-targeting RGD component and a DNA-binding moiety consisting of 16 lysine residues. The peptide binds to cell surface integrins, forms distinct, protective complexes with DNA, and can deliver DNA to cells for transgene expression. The level of reporter gene expression obtained is dependent on the [K]₁₆RGD-to-DNA ratio and is enhanced in the presence of the endosomal buffer chloroquine or lysosomal disrupting agents during gene delivery. Levels of reporter gene expression obtained under ideal conditions with [K]₁₆RGD approach or even exceed those obtained with DNA delivered by Lipofectamine.