Intracellular distribution of microinjected antisense oligonucleotides.

Abstract

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...

Effect of unmodified triple helix‐forming oligodeoxyribonucleotide targeted to human multidrug‐resistance gene mdr1 in MDR cancer cells

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Anti-inflammatory gene therapy directed at the airway epithelium

“…Cell division may not be required for intranuclear uptake of oligonucleotides, since they enter the nucleus rapidly and their accumulation is temperature and energy-independent. 29,32 In contrast, plasmid uptake into the nucleus appears to be enhanced through cell division 33 and this process is energy-and temperature-dependent. 34 In contrast to I B␣ overexpression, decoys do not require protein expression, a further potential advantage.…”