Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis.
Nucleic Acids Res
; 36(8): 2764-76, 2008 May.
Article
en En
| MEDLINE
| ID: mdl-18367474
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 alphavbeta3 integrin. We used alphavbeta3-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 alphavbeta3 integrin.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Oligopéptidos
/
Oligonucleótidos Antisentido
/
Integrina alfaVbeta3
/
Endocitosis
Límite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2008
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Reino Unido