Variations in mRNA content have no effect on the potency of antisense oligonucleotides

A fundamental question with regard to antisense pharmacology is the extent to which RNA content or transcription rate or both affect the potency of an tisense drugs. We have addressed this by controlling RNA content and transc ription rate using either an exogenous gene expressed after transfection or an endogenous gene induced with a cytokine. We have demonstrated that in b oth A549 and HeLa cells, varying RNA copy numbers from <1 to >100 copies pe r cell has no effect on the potency of RNase H-active antisense drugs trans fected into cells, nor did variation in transcription rate have an effect o n potency. We demonstrate that this is because the number of oligonucleotid e molecules per cell is vastly in excess of the RNA copy number. These data further suggest that a significant fraction of cell-associated antisense d rug molecules may be unavailable to interact with the target RNA, an observ ation that is not surprising, as phosphorothioate oligonucleotides interact with many cellular proteins. We suggest that these data may extrapolate to in vivo results.