ABSTRACT
The fact that antisense Oligonucleotides can reach target mRNAs within cells is really quite remarkable from a membrane transport perspective. Cell membranes are designed by nature to be very impermeable to large, polar molecules such as Oligonucleotides, unless specific transport pathways are available. For charged Oligonucleotides, there is good evidence that initial accumulation in cells is largely due to some form of endocytosis (Akhtar and Juliano, 1992; Bennett, 1998; Stein and Cheng, 1993; Wagner, 1994). Studies using fluorescent conjugates (Beltinger et al., 1995; Shoji et al., 1991; Stein and Cheng, 1993; Wagner, 1994) have shown that Oligonucleotides taken up by intact cells have a punctate cytoplasmic distribution, and are initially localized in cytoplasmic vesicles that are probably endosomes; in most cases little fluorescence is associated with the nucleus. However, when fluorescent Oligonucleotides are microinjected directly into the cytoplasm they rapidly redistribute to the nucleus (Fisher et al., 1993; Leonetti et al., 1991). Therefore, intact Oligonucleotides leaking out of endosomes would be expected to accumulate rapidly in the nucleus, but this is not seen in most cells, indicating very limited transfer of Oligonucleotides from endosomal to cytoplasmic compartments. However, since antisense effects clearly do occur, some transfer must take place. Oligonucleotides not only accumulate in endosomes, but they can also recycle back to the cell surface and be released; this seems to be particularly true for material accumulating in so-called ‘shallow’ endosomal compartments (Stein and Cheng, 1993; see also Chapter 9). Although many cells take up Oligonucleotides via endocytosis, some interesting recent studies have suggested that the endocytotic rates for Oligonucleotides are different for the apical and basolateral regions of polarized epithelial cells (Takakura et al., 1998).
