705. A Practical Approach to siRNA-Based Treatment Design Using Bioluminescent Imaging and Mathematical Modeling

Abstract

Small interfering RNA (siRNA) molecules achieve sequence-specific gene silencing through a process known as RNA interference (RNAi). Compared to other nucleic acid-based therapeutics aimed at post-transcriptional gene silencing, such as antisense oligodeoxynucleotides, siRNA molecules achieve greater magnitude and duration of gene silencing at significantly lower doses. While the duration of gene knockdown by siRNA typically lasts around 1 week in rapidly dividing cells, recent reports of knockdown lasting for several weeks in nondividing cells indicate that dilution due to cell division may be a limiting factor in rapidly dividing cells. To determine if cell division directly impacts the duration of gene knockdown by siRNA, we chose to investigate the kinetics of siRNA-mediated gene silencing in luciferase-expressing cell lines with different observed doubling times using noninvasive bioluminescent imaging and a mathematical model of siRNA delivery and function. In vitro and in vivo, the duration of gene knockdown is inversely proportional to the rate of cell division. Consistent with previous reports, luciferase protein levels recover to pre-treatment values within less than 1 week in rapidly dividing cell lines, but take longer than 3 weeks to return to steady-state levels in nondividing fibroblasts. Similar results are observed in vivo, with knockdown lasting around 1 week in subcutaneous tumors in A/J mice and 3-4 weeks in the nondividing hepatocytes of BALB/c mice. These data indicate that dilution due to cell division, and not intracellular siRNA half-life, governs the duration of gene silencing under these conditions. Here, we will present our latest results describing the effects of cell doubling time, siRNA stability, and dosing schedule on siRNA- mediated gene silencing. Specifically, we will investigate whether the duration of knockdown using chemically modified siRNA molecules exhibits a similar dependence on cell doubling time. The implications of these findings will be highlighted using model calculations to determine the dosing schedule required to maintain persistent silencing of target proteins and to predict when maximum mRNA or protein knockdown will occur, an especially important factor when trying to observe a therapeutic effect resulting from protein knockdown. The approach of bioluminescent imaging combined with mathematical modeling provides insights into siRNA function that will hopefully be of practical use for both researchers and clinicians alike.