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 intracellu lar transport to RNA target. To overcome these obstacles, ODN carriers have been developed, but the intracellular fate of ODNs is controversial and st rongly depends on die means of vectorization. Polyamidoamine dendrimers are non-linear polycationic cascade polymers that are able to bind ODNs electr ostatically. These complexes have been demonstrated to protect phosphodiest er ODNs from nuclease degradation and also to increase their cellular uptak e and pharmacological effectiveness. We studied the intracellular distribut ion of a fluorescein isothiocyanate-labeled ODN vectorized by a dendrimer v ector and found that intracellular ODN distribution was dependent on the ph ase 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 repor ter genes, one used as target, the other as control and vice versa. This sy stem 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. BIOCHEM PHARMACOL 58;1:95-10 7, 1999. (C) 1999 Elsevier Science Inc.