Enhanced downregulation of the p75 nerve growth factor receptor by cholesteryl and bis-cholesteryl antisense oligonucleotides

The effects of conjugating cholesterol to either or both ends of a phosphor othioate (PS) oligonucleotide were analyzed in terms of cellular uptake and antisense efficacy. The oligo sequence was directed against the p75 nerve growth factor receptor (p75), and was tested in differentiated PC12 cells, which express high levels of this protein. The addition of a single cholest eryl group to the 5'-end significantly increased cellular uptake and improv ed p75 mRNA downregulation compared with the unmodified PS oligo, However, only a minor degree of downregulation of p75 protein was obtained with 5' c holesteryl oligos, Three different linkers was used to attach the 5' choles teryl group but were found not to have any impact on efficacy. Addition of a single cholesteryl group to the 3'-end led to greater p75 mRNA downregula tion (31%) and p75 protein downregulation (28%) than occurred with the 5' c holesteryl oligos. The biggest improvement in antisense efficacy, both at t he mRNA and protein levels, was obtained from the conjugation of cholestero l to both ends of the oligo. One of the bis-cholesteryl oligos was nearly a s effective as cycloheximide at decreasing synthesis of p75, The bis-choles teryl oligos also displayed significant efficacy at 1 mu M, whereas the oth er oligos required 5 mu M to be effective. The enhanced efficacy of bis-cho lesteryl oligos is likely to be due to a combination of enhanced cellular u ptake and resistance to both 5' and 3' exonucleases.