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The distribution of intravenously
injected phosphorothioate oligodeoxynucleotides (P = S ODN) was studied
in vivo in rodent tissues using three histologic methods: immunohistochemistry
with a monoclonal antibody that recognizes P = S ODN ISIS 2105; direct fluorescence
microscopy of P = S ODN ISIS 2105 conjugated to rhodamine; and autoradiography
of 14C-labeled P = S ODN ISIS 2302. All three methods gave the same pattern
of oligonucleotide distribution, and the intensity of the histologic signal
agreed with previously published pharmacokinetic data on the relative concentration
of P = S ODN in different organs. Proximal tubule cells in the kidney and
Kupffer and endothelial cells in the liver 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 the skin, were also labeled, whereas the P = S
ODN signal was weak or negative in epithelial and muscle cells in the skin
and intestine. At 2 hours postinjection, P = S ODN were clearly detectable
in the extracellular matrix in loose and dense connective tissues, although
by 24 hours, the label was predominantly intracellular. Large, nucleated
cells in red marrow, and the connective tissues around bone and skeletal
muscle cells and lining the knee joint, were positive for oligonucleotide,
whereas P = S ODN were not detected in erythrocytes, cartilage, compact
bone, and skeletal muscle. In spleen, white pulp was negative for P = S
ODN, whereas cells surrounding the sinusoids and nucleated cells in the
red pulp were strongly positive for P = S ODN. Our results provide 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 and the distribution of modified
oligonucleotides. Furthermore, because our results indicate which cell types
are likely to be affected by antisense oligonuclotides, they can be used
to guide future in vivo applications of the technology. |
