Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis

We describe the synthesis and characterization of a 5 conjugate between a 2-O-Me phosphorothioate antisense oligonucleotide and a bivalent RGD (arginineglycineaspartic acid) peptide that is a high-affinity ligand for the alpha v beta 3 integrin. We used alpha v beta 3-positive melanoma cells transfected with a reporter comprised of the firefly luciferase gene interrupted by an abnormally spliced intron. Intranuclear delivery of a specific antisense oligonucleotide (termed 623) corrects splicing and allows luciferase expression in these cells. The RGD623 conjugate or a cationic lipid-623 complex produced significant increases in luciferase expression, while free 623 did not. However, the kinetics of luciferase expression was distinct; the RGD623 conjugate produced a gradual increase followed by a gradual decline, while the cationic lipid-623 complex caused a rapid increase followed by a monotonic decline. The subcellular distribution of the oligonucleotide delivered using cationic lipids included both cytoplasmic vesicles and the nucleus, while the RGD623 conjugate was primarily found in cytoplasmic vesicles that partially co-localized with a marker for caveolae. Both the cellular uptake and the biological effect of the RGD623 conjugate were blocked by excess RGD peptide. These observations suggest that the bivalent RGD peptideoligonucleotide conjugate enters cells via a process of receptor-mediated endocytosis mediated by the alpha v beta 3 integrin.