Binding of antisense phosphorothioate oligonucleotides to murine lymphocytes is lineage specific and inducible.
A phosphorothioate oligonucleotide that has been employed to inhibit HIV-1 viral expression in chronically infected H9 cells was examined for its ability to associate with murine lymphoid cells. The relationship between cellular oligonucleotide concentration and the lymphoid target tissues is important to the selection of an animal model, evaluation of potential side effects, and understanding the actions of a therapeutically useful antisense oligonucleotide. Lymphoid cells were harvested from murine peripheral blood, bone marrow, thymus, lymph node, and spleen. Cell subpopulations that bind the oligonucleotide were distinguished by two-color flow cytometry employing a fluorescein-labeled anti-rev oligonucleotide and phycoerythrin-labeled antibodies to selected cell surface molecules associated with unique subpopulations of cells. Very little oligonucleotide binding was observed in peripheral blood mononuclear cells or thymic T cells, but substantial numbers of cells, primarily B cells from bone marrow and spleen, accumulated the oligonucleotide. The cell-associated oligonucleotide was increased significantly in lymphoid populations when the cells were mitogen pretreated with either concanavalin-A (ConA), a T cell mitogen, or lipopolysaccharide (LPS), a B cell mitogen. These data clearly demonstrate the ability of fluorescein-conjugated oligonucleotides to bind to unique cell populations in suspension, allowing simultaneous two-color phenotypic analysis, suggesting that fluorescein-conjugated oligonucleotides may be a useful bridge between in vitro molecular biology techniques and in vivo cell biology. In addition, these data provide optimism concerning the in vivo treatment of chronically infected HIV patients using antisense oligonucleotides.
Iversen
P L
PL
Department of Pharmacology, University of Nebraska Medical Center, Omaha.
Crouse
D
D
Zon
G
G
Perry
G
G
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001853:Bone Marrow / Q000378:metabolism; D001854:Bone Marrow Cells; D005260:Female; D008198:Lymph Nodes / Q000166:cytology / Q000378:metabolism; D008213:Lymphocyte Activation; D016131:Lymphocyte Subsets / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism*; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism
D016376:Oligonucleotides, Antisense; D013873:Thionucleotides