Optimization of functional efficacy of phosphorothioate-modified oligonucleotides in a human CD8+ T-cell ex vivo expansion model.
Antisense oligodeoxyribonucleotides (ODNs) can specifically inhibit gene expression, but their application to fresh human CD8+ T cells is limited by poor spontaneous uptake (<2%). We have examined and optimized the uptake of phosphorothioate-modified oligodeoxyribonucleotides (PS-ODNs) into these cells in an ex vivo expansion model. Optimal antisense treatments were found to be, for fresh CD8+ T cells, 1 micro m PS-ODNs complexed with lipofectin (LF), which resulted in 35% uptake and 10 micro m PS-ODNs in the absence of LF, for cultured cells, which resulted in 95% uptake. The delivered antisenses were functional, as determined by the inhibition of protein expression. In this respect, partially phosphorothioate-modified ODNs (PS-ODNs-P) were twice as effective as completely modified (PS-ODNs-C), and the antisense specific for the cap site showed the highest protein suppression of those tested (68%). Uptake mechanisms were also investigated. To our knowledge, this is the first optimization of the delivery of antisense oligonucleotides into human CD8+ T cells. This protocol could be used to study the function of a particular gene in cytotoxic T lymphocytes and also by those looking for a method to deliver short interfering RNA into cell lines to specifically suppress a gene of interest.
D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology*; D006801:Humans; D009841:Oligonucleotides / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism
D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D011509:Proteoglycans