Antisense oligomers constitute an attractive class of specific tools for genetic analysis and for potential therapeutic applications. Targets with different cellular locations have been described, such as mRNA translation initiation sites, pre-mRNA splicing sites, or the genes themselves. However the mechanism(s) of action and the intracellular distribution of antisense oligomers remain poorly understood. Antisense oligomers conjugated with various fluorochromes or with BrdUrd were microinected into the cytoplasm of somatic cells, and their cellular distribution was monitored by fluorescence microscopy in fixed and nonfixed cells. A fast translocation in the nuclei and a concentration on nuclear structures were observed whatever probe was used. Nuclear transport occurs by diffusion since it is not affected by depletion of the intracellular ATP pool, temperature, or excess unlabeled oligomer. Accumulation of the oligomers in the nuclei essentially takes place on a set of proteins preferentially extracted between 0.2 M and 0.4 M NaCI as revealed by crosslinking of photosensitive oligomers. The relationship between nuclear location of antisense oligomers and their mechanism of action remains to be ascertained and could be of major interest in the design of more efficient antisense molecules.The ability of antisense oligomers to interfere specifically with mRNAs or their precursors provides a valuable tool for the control of cellular and viral gene expression (for review, see ref. 1). Although attractive, this approach is restricted by the poor stability of natural oligomers and the low efficiency of their cellular uptake. These difficulties can at least be partially circumvented by the use of nuclease-resistant analogs or drug delivery techniques (2). Despite these advances, the design of optimal strategies for oligomer synthesis and delivery has remained difficult since not much is known about the mechanism through which antisense oligomers interfere with gene expression within intact cells or of their intracellular fate.The antisense concept is based on the assumption that complementary DNA or RNA sequences might interact with mRNA to block their translation. To overcome the complexity of investigation in intact cells, several laboratories have developed cell-free models to evaluate the inhibitory potential of these molecules. Nevertheless, cell-free studies have usually been limited to observations at the translational level and are not always representative of the intact cell situation. Antisense oligomers were successfully used on cultured cells against various targets, such as translation initiation (3), introns (4), and splice sites (5). The subcellular locations of these targets lead to the assumption that oligomers might act in the cytoplasm and/or the nuclei.Charged oligomers are internalized through an endocytic pathway (6, 7), and a punctuated cytoplasmic labeling characteristic of accumulation in the endocytic vesicles is indeed observed when fluorescently tagged oligomers are used (6). However...

We have previously shown that antisense oligomers linked to poly(L-lysine) (PLL) exhibit antiviral properties against vesicular stomatitis virus (VSV) at concentrations lower than 1 microM. The conjugation to PLL provides an interesting alternative to natural or neutral oligomers to increase the biological effects of antisense oligomers. The internalization pathway of oligomer-PLL conjugates as compared to unconjugated oligomers has been studied in L929 cells. In parallel to their enhanced antiviral activity, PLL increases greatly the uptake of fluorescently tagged oligomers. This internalization follows a classical endocytic pathway and the oligomer has to be cleaved from PLL in the cell to exhibit an antiviral effect.

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