IntroductionApplication of antisense deoxynucleotides (ODN) may constitute an alternative clinical approach for specifically regulating the expression of genes. However, the therapeutic activity of ODN molecules is often impaired by their susceptibility to untimely metabolic inactivation and by their inability to cross the cellular membrane. A promising strategy to circumvent both problems involves specific targeting of ODN to the aimed cell type. In this study, we have studied the in vivo fate of 3 2 P-labeled ODN in rats in order to assess whether ODN itself is prone to tissue-specific accumulation.Methods and Results 3 2 P-ODN was rapidly cleared from the bloodstream upon intravenous injection into rats. Approximately 45% of the injected dose of 3 2 P-ODN was recovered in the liver within 5 min after injection. Non-parenchymal liver cells, and Kupffer cells in particular, were responsible for the hepatic uptake of 3 2 P-ODN. Hepatic uptake of 3 2 P-ODN appeared to be dependent on the tertiary structure of the ODN, as liver uptake of 3 2 P-ODN was greatly impaired after heat-induced denaturation. To elucidate the molecular basis of the liver uptake of 3 2 P-ODN, we have studied the interaction of 3 2 P-ODN with isolated rat endothelial (EC) and Kupffer cells (KC) using in vitro binding and uptake studies. 3 2 P-ODN binding to EC and KC was saturable (50.3 and 26.7 ng/mg, respectively) and of moderate affinity (Kd=51 and 117 nM, respectively). Binding was almost abolished by preincubating the cells with trypsine (37°C, 15 min) and after an acid wash (pH 2.8), indicating that binding is mediated by a proteidic recognition site. Binding to EC and KC could be inhibited for 90% by ODN, polyinosinic acid, fucoidin and nucleotide di- and triphosphates, whereas heat-denaturated ODN, polycytidinic acid, polyadenosinic acid, ssDNA, and nucleotide or nucleotide monophosphate analogues were completely ineffective. Surprisingly, acetylated low-density-lipoprotein, an established substrate for the scavenger receptor on endothelial and Kupffer cells, was not able to inhibit 3 2 P-ODN binding.Uptake studies at 37°C demonstrated that ligand binding was efficiently coupled to internalisation and lysosomal processing. Moreover, intact ODN could still be recovered in the lysosomal compartment after incubation of EC and KC with 3 2 P-ODN.ConclusionsIt can be concluded that endothelial and Kupffer cells possess ODN-specific recognition sites. The inhibition profile of 3 2 P-ODN binding sites on both cell types is essentially equal; and corresponds closely to that of the scavenger receptor. The fact that, in vivo, 3 2 P-ODN is mainly transported to non-parenchymal liver cells and taken up via receptor-mediated endocytosis may have implications for potential in vivo application of antisense ODN in the regulation of gene expression of endothelial and Kupffer cell-specific gene products. Leiden University, Leiden Institute of Chemistry, Gorlaeus Laboratories.