Nucleic Acids Research Advance Access published online on March 26, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn115
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Molecular Biology |
Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis
1Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill NC 27599 and 2Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305-5484, USA
*To whom correspondence should be addressed. Tel: 919 966 4383; Fax: 919 966 5640; Email: arjay{at}med.unc.edu
Received January 14, 2008. Revised February 14, 2008. Accepted February 19, 2008.
We describe the synthesis and characterization of a 5' conjugate between a 2'-O-Me phosphorothioate antisense oligonucleotide and a bivalent RGD (arginine–glycine–aspartic acid) peptide that is a high-affinity ligand for the 
vβ3 integrin. We used vβ3-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 RGD–623 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 RGD–623 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 RGD–623 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 RGD–623 conjugate were blocked by excess RGD peptide. These observations suggest that the bivalent RGD peptide–oligonucleotide conjugate enters cells via a process of receptor-mediated endocytosis mediated by the vβ3 integrin.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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