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.

Liposomes enhance delivery and expression of an RGD-oligolysine gene transfer vector in human tracheal cells

“…12 16 RGD/DNA ratio without liposome and is about sixfold (P Ͻ 0.01) higher than that of [K] 16 /DNA in the presence of lipofectamine (result not shown). At this liposome ratio (24 g/ g DNA) it appears that lipofectamine masks the RGD sequence making it less accessible to cell surface integrins and that transfection is mainly driven by the nonspecific mechanism of liposome-mediated cell entry.…”

“…We demonstrated previously by competitive cell binding studies with the [K] 16 RGD peptide that the RGD domain in our construct maintained its affinity for these integrins after the synthetic fusion with the highly positively charged oligo-llysine chain. 12 These experiments were performed on the Caco-2 intestinal cell line which our group recently showed to express the ␤1-integrin subunit. 22 Since the present study uses tracheal epithelial cells with the longterm aim of correcting the cystic fibrosis defect in airway epithelium, we verified that this cell line also demonstrates binding affinity for the RGD domain of the [K] 16 16 RGD competes with FN or VN for attachment of the epithelial 56FHTE8o − cell line and suggests that these cells express integrins which recognise the RGD motif.…”

“…This nonviral vector system is a first step towards development of cell-specific targeting of the airway for gene therapy of cystic fibrosis. 12 Peptides were purified by HPLC on a C18 preparative column and characterised by matrix-assisted laser desorption/ionisationtime of flight (MALDI-TOF) mass spectrometry and amino acid sequencing, as described previously. 12 The purity of each peptide was in excess of 95%.…”

“…This molecule is comprised of an integrin receptor-targeting domain containing an arginine-glycine-aspartic acid tripeptide motif and a DNA binding moiety consisting of a stretch of 16 lysine residues, Arg-GlyAsp/oligo-l-lysine ([K] 16 RGD). 11,12 We showed that this peptide condensed plasmid DNA (approximately 10 nm) rendering it resistant to nuclease degradation and that the level of reporter gene expression obtained in Caco-2 cells under optimal conditions approached that of the commercial liposome lipofectamine. 12 Integrins are a class of related heterodimeric transmembrane surface receptors involved in cell-cell adhesion and in promotion of interactions between cells and components of the extracellular matrix (for review see Ref.…”

Glycosylation-mediated targeting of carriers

“…So far, galactose, mannose, fucose, mannose-6-phosphate, sialyl Lewis x, peptides, and proteins (i.e., transferrin, epidermal growth factor, and anti-human epidermal growth factor receptor 2 antibodies) have been used as ligands [3][4][5][6][7][8][9][10]. Among the various ligands reported, sugar recognition mechanisms are a promising approach for active targeting because of their high affinity and expression.…”