Delivery of antisense oligonucleotides to target cells-a done deal?

摘要

Antisense oligonucleotides have been employed in literally hundreds of experiments in an attempt to specifically down regulate gene expression. Phosphodiester (normal) DNA cannot, however, be used for these types of experiments because it is degraded by ubiquitous DNases, which appear to be predominately exonucleolytic. Over the past several decades, chemists have synthesized a variety of nuclease resistant oligodeoxynucleotides, of which the most commonly available are the phosphorothioates. These molecules contain a sulfur atom that has been substituted for a non-bridging oxygen atom at every phosphorus in the oligonucleotide chain. Phosphorothioate oligodeoxynucleotides have been shown in tissue culture to be able to specifically down regulate the expression of a large number of genes, but their usage is complicated by the fact that they bind with very high affinity to heparin-binding proteins. In addition, because they are polyanions, they cannot passively diffuse across cell membranes, and when internalized, via the processes of adsorptive endocytosis and fluid phase endocytosis, they come to reside in the cellular vesicular system (i.e., endosomes and lysosomes). The spontaneous endosomal rupture rate in at least most cell types appears to be sufficiently slow to preclude nuclear concentrations high enough for the production of antisense effects.