CELLULAR-DISTRIBUTION OF PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDES IN NORMAL RODENT TISSUES

The distribution of intravenously injected phosphorothioate oligodeoxy nucleotides (P = S ODN) was studied in vivo in rodent tissues using th ree histologic methods: immunohistochemistry with a monoclonal antibod y that recognizes P = S ODN ISIS 2105; direct fluorescence microscopy of P = S ODN ISIS 2105 conjugated to rhodamine; and autoradiography of C-14-labeled P = S ODN ISIS 2302. All three methods gave the same pat tern of oligonucleotide distribution, and the intensity of the histolo gic signal agreed with previously published pharmacokinetic data on th e relative concentration of P = S ODN in different organs. Proximal tu bule cells in the kidney and Kupffer and endothelial cells in the live r were among the most heavily labeled with P = S ODN at all doses and time-points. Connective tissues proper, such as the lamina propria and submucosa of the intestine and the dermis and subcutaneous layer of t he skin, were also labeled, whereas the P = S ODN signal was weak or n egative in epithelial and muscle cells in the skin and intestine. At 2 hours postinjection, P = S ODN were clearly detectable in the extrace llular matrix in loose and dense connective tissues, although by 24 ho urs, the label was predominantly intracellular. Large, nucleated cells in red marrow, and the connective tissues around bone and skeletal mu scle cells and lining the knee joint, were positive for oligonucleotid e, whereas P = S ODN were not detected in erythrocytes, cartilage, com pact bone, and skeletal muscle. In spleen, white pulp was negative for P = S ODN, whereas cells surrounding the sinusoids and nucleated cell s in the red pulp were strongly positive for P = S ODN. Our results pr ovide specific information on the tissue and cellular localization of P = S ODN within organs in vivo. The data presented will be used as a reference for studies of P = S ODN distribution in diseased tissues an d the distribution of modified oligonucleotides. Furthermore, because our results indicate which cell types are likely to be affected by ant isense oligonucleotides, they can be used to guide future in vivo appl ications of the technology.