Optimization of Functional Efficacy of Phosphorothioate‐Modified Oligonucleotides in a Human CD8⁺ T‐Cell Ex Vivo Expansion Model

Abstract

Antisense oligodeoxyribonucleotides (ODNs) can specifically inhibit gene expression, but their application to fresh human CD8 þ 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 þ T cells, 1 mM PS-ODNs complexed with lipofectin (LF), which resulted in 35% uptake and 10 mM 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 þ 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.

Investigation of alpha nascent polypeptide‐associated complex functions in a human CD8+ T cell ex vivo expansion model using antisense oligonucleotides

“…Recently, we have optimized the conditions affecting the functional efficacy of PS‐ODNs 20 . In this study, anti‐cap PS‐ODN partially phosphorothioate modified antisense was used to investigate the biological functions of the 0·8‐kb α NAC gene in human CD8 + T cells as it showed optimal inhibition of the α NAC protein 20 . CD8 + T cells in each of the two culture conditions were treated with different PS‐ODNs.…”

“…20 In this study, anti-cap PS-ODN partially phosphorothioate modified antisense was used to investigate the biological functions of the 0AE8kb a NAC gene in human CD8 + T cells as it showed optimal inhibition of the a NAC protein. 20 CD8 + T cells in each of the two culture conditions were treated with different PS-ODNs. The two blots were densiometrically scanned to quantify the percentage inhibition of a NAC protein expression.…”