Oligodeoxyribonucleotide uptake in primary human hematopoietic cells is enhanced by cationic lipids and depends on the hematopoietic cell subset.
The use of antisense oligodeoxyribonucleotides (ODN) is a potential method to switch off gene expression. The poor cellular uptake of ODN in primary cells still is a limiting factor that may contribute to the lack of functional efficacy. Various forms of cationic lipids have been developed for efficient delivery of nucleic acids into different cell types. We examined the two cationic lipids DOTAP and DOSPER to improve uptake of ODN into primary human hematopoietic cells. Using a radiolabeled 23-mer, ODN uptake into blood-derived mononuclear cells could be increased 42- to 93-fold by DOTAP and 440- to 1,025-fold by DOSPER compared with application of ODN alone. DOTAP was also effective for delivery of ODN into leukocytes within whole blood, which may resemble more closely the in vivo conditions. As assessed by fluorescein isothiocyanate-conjugated ODN both cationic lipids enhanced cytoplasmic accumulation of ODN in endosome/lysosome-like structures with a partial shift of fluorescence to the whole cytoplasm and the nucleus following an incubation of 24 hours. ODN uptake by cationic lipids into different hematopoietic cell subsets was examined by dual-color immunofluorescence analysis with subset-specific monoclonal antibodies. We found a cell type-dependent delivery of ODN with greatest uptake in monocytes and smallest uptake in T cells. CD34+ cells, B cells, and granulocytes took up ODN at an intermediate level. Uptake of ODN into isolated CD34+ cells could be increased 100- to 240-fold using cationic lipids compared with application of ODN alone. Stimulation of CD34+ cells by interleukin-3 (IL-3), IL-6, and stem cell factor did not significantly improve cationic lipid-mediated ODN delivery. Sequence-specific antisense effects in clonogenic assays could be shown by transfection of bcr-abl oncogene-directed antisense ODN into primary cells of patients with chronic myelogenous leukemia using this established protocol. In conclusion, cationic lipids may be useful tools for delivery of antisense ODN into primary hematopoietic cells. These studies provide a basis for clinical protocols in the treatment of hematopoietic cells in patients with hematologic malignancies and viral diseases by antisense ODN.
Kronenwett
R
R
Klinische Kooperationseinheit Molekulare Hämatologie/Onkologie and Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Steidl
U
U
Kirsch
M
M
Sczakiel
G
G
Haas
R
R
D000911:Antibodies, Monoclonal; D018952:Antigens, CD34 / Q000032:analysis; D001402:B-Lymphocytes / Q000378:metabolism; D002412:Cations; D005455:Fluorescent Antibody Technique; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D006098:Granulocytes / Q000378:metabolism; D019337:Hematologic Neoplasms / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism; D008055:Lipids / Q000494:pharmacology*; D009000:Monocytes / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D018952:Antigens, CD34; D002412:Cations; D007377:Interleukin-3; D008055:Lipids; D016376:Oligonucleotides, Antisense; D016044:Fusion Proteins, bcr-abl