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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 (arginine–glycine–aspartic acid) peptide that is a high-affinity ligand for the αvβ3 integrin. We used αvβ3-positive melanoma cells transfected with a repo...

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Autores principales: Alam, Md Rowshon, Dixit, Vidula, Kang, Hyunmin, Li, Zi-Bo, Chen, Xiaoyuan, Trejo, JoAnn, Fisher, Michael, Juliano, Rudy L.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2377441/
https://www.ncbi.nlm.nih.gov/pubmed/18367474
http://dx.doi.org/10.1093/nar/gkn115
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author Alam, Md Rowshon
Dixit, Vidula
Kang, Hyunmin
Li, Zi-Bo
Chen, Xiaoyuan
Trejo, JoAnn
Fisher, Michael
Juliano, Rudy L.
author_facet Alam, Md Rowshon
Dixit, Vidula
Kang, Hyunmin
Li, Zi-Bo
Chen, Xiaoyuan
Trejo, JoAnn
Fisher, Michael
Juliano, Rudy L.
author_sort Alam, Md Rowshon
collection PubMed
description 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 αvβ3 integrin. We used αvβ3-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 αvβ3 integrin.
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spelling pubmed-23774412008-05-14 Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis Alam, Md Rowshon Dixit, Vidula Kang, Hyunmin Li, Zi-Bo Chen, Xiaoyuan Trejo, JoAnn Fisher, Michael Juliano, Rudy L. Nucleic Acids Res Molecular Biology 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 αvβ3 integrin. We used αvβ3-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 αvβ3 integrin. Oxford University Press 2008-05 2008-03-26 /pmc/articles/PMC2377441/ /pubmed/18367474 http://dx.doi.org/10.1093/nar/gkn115 Text en © 2008 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Molecular Biology
Alam, Md Rowshon
Dixit, Vidula
Kang, Hyunmin
Li, Zi-Bo
Chen, Xiaoyuan
Trejo, JoAnn
Fisher, Michael
Juliano, Rudy L.
Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis
title Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis
title_full Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis
title_fullStr Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis
title_full_unstemmed Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis
title_short Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis
title_sort intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis
topic Molecular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2377441/
https://www.ncbi.nlm.nih.gov/pubmed/18367474
http://dx.doi.org/10.1093/nar/gkn115
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