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Optimization of Functional Efficacy of Phosphorothioate‐Modified Oligonucleotides in a Human CD8 + T‐Cell Ex Vivo Expansion Model
Author(s) -
AlShanti N. A.,
Steward C. G.,
Garland R. J.
Publication year - 2003
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1046/j.1365-3083.2003.01319.x
Subject(s) - ex vivo , cytotoxic t cell , microbiology and biotechnology , oligonucleotide , cd8 , in vivo , in vitro , biology , gene expression , cell , t cell , gene , chemistry , biochemistry , immune system , immunology , genetics
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 µ m PS‐ODNs complexed with lipofectin (LF), which resulted in 35% uptake and 10 µ 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.