Cell cycle-dependent distribution and specific inhibitory effect of vectorized antisense oligonucleotides in cell culture.
Factors limiting the use of antisense phosphodiester oligodeoxynucleotides (ODNs) as therapeutic agents are inefficient cellular uptake and intracellular transport to RNA target. To overcome these obstacles, ODN carriers have been developed, but the intracellular fate of ODNs is controversial and strongly depends on the means of vectorization. Polyamidoamine dendrimers are non-linear polycationic cascade polymers that are able to bind ODNs electrostatically. These complexes have been demonstrated to protect phosphodiester ODNs from nuclease degradation and also to increase their cellular uptake and pharmacological effectiveness. We studied the intracellular distribution of a fluorescein isothiocyanate-labeled ODN vectorized by a dendrimer vector and found that intracellular ODN distribution was dependent on the phase of the cell cycle, with a nuclear localization predominantly in the G2/M phase. In addition, in order to evaluate the relevance of ODN vectors in enhancing the inhibition of the targeted genes' expression, we developed a rapid screening system which measures the transient expression of two reporter genes, one used as target, the other as control and vice versa. This system was validated through investigating the effect of the dendrimer vector on ODN biological activity. Antisense sequence-specific inhibition of more than 70% of one reporter gene was obtained with a chimeric ODN containing four phosphorothioate groups, two at each end.
Hélin
V
V
Laboratoire de Biochimie-Enzymologie, UMR 8532, Institut Gustave-Roussy, Villejuif, France. vhelin@igr.fr
Gottikh
M
M
Mishal
Z
Z
Subra
F
F
Malvy
C
C
Lavignon
M
M
D016475:3T3 Cells; D000818:Animals; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000378:metabolism / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D018613:Microscopy, Confocal; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis
D008164:Luminescent Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins