Rational selection and quantitative evaluation of antisense oligonucleotides

Antisense oligonucleotides are an attractive therapeutic option to modulate specific gene expression. However, not all antisense oligonucleotides are effective in inhibiting gene expression, and currently very few methods exi st for selecting the few effective ones from all candidate oligonucleotides . The lack of quantitative methods to rapidly assess the efficacy of antise nse oligonucleotides also contributes to the difficulty of discovering pote nt and specific antisense oligonucleotides. We have previously reported the development of a prediction algorithm for identifying high affinity antise nse oligonucleotides based on mRNA-oligonucleotide hybridization. In this s tudy, we report the antisense activity of these rationally selected oligonu cleotides against three model target mRNAs (human lactate dehydrogenase A a nd B and rat gp130) in cell culture. The effectiveness of oligonuclectides was evaluated by a kinetic PCR technique, which allows quantitative evaluat ion of mRNA levels and thus provides a measure of antisense-mediated decrea ses in target mRNA, as occurs through RNase H recruitment. Antisense oligon ucleotides that were predicted to have high affinity for their target prove d effective in almost all cases, including tests against three different ta rgets in two cell types with phosphodiester and phosphorothioate oligonucle otide chemistries. This approach may aid the development of antisense oligo nucleotides for a variety of applications. (C) 2001 Elsevier Science B.V. A ll rights reserved.