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. 2006 Jan 12;34(1):322-33.
doi: 10.1093/nar/gkj439. Print 2006.

Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging

Derek W Bartlett  1 , Mark E Davis
Affiliations

Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging

Derek W Bartlett et al. Nucleic Acids Res. .

Abstract

Small interfering RNA (siRNA) molecules are potent effectors of post-transcriptional gene silencing. Using noninvasive bioluminescent imaging and a mathematical model of siRNA delivery and function, the effects of target-specific and treatment-specific parameters on siRNA-mediated gene silencing are monitored in cells stably expressing the firefly luciferase protein. In vitro, luciferase protein levels recover to pre-treatment values within <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 approximately 10 days 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. To demonstrate the practical use of the model in treatment design, model calculations are used to predict the dosing schedule required to maintain persistent silencing of target proteins with different half-lives in rapidly dividing or nondividing cells. The approach of bioluminescent imaging combined with mathematical modeling provides useful insights into siRNA function and may help expedite the translation of siRNA into clinically relevant therapeutics for disease treatment and management.

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Figures

Figure 1
Figure 1
Simplified schematic of the key steps required for siRNA delivery to and function within mammalian cells. Steps 1–3 are unique to in vivo application of siRNA, whereas steps 4–9 represent the general processes on the level of an individual cell and are therefore common to both in vivo and in vitro application of siRNA.
Figure 2
Figure 2
Effect of siRNA dose on the duration and magnitude of luciferase knockdown by siRNA in nondividing cells. (A) Experimental results using Oligofectamine to deliver siRNA to luciferase-expressing, nondividing fibroblasts with 1.5 × 105 cells per well in a 24-well plate. Data points represent the ratio of the average luciferase signal intensity from triplicate wells receiving siGL3 and siCON1 on day 0. Squares, 10 nM; diamonds, 25 nM; triangles, 50 nM; circles, 100 nM. (B) Luciferase knockdown after siRNA transfection predicted by the mathematical model using the baseline in vitro parameters given in Table 2 with the number of cells held constant at 1.5 × 105, a transfection time of 5 h, and a transfection efficiency of 90%.
Figure 3
Figure 3
Effect of cell doubling time on the duration of luciferase knockdown by siRNA in vitro. (A) Experimental results using Oligofectamine to deliver 100 nM siRNA to luciferase-expressing cells with a range of doubling times (dt). Data points represent the ratio of the average luciferase signal intensity from triplicate wells receiving siGL3 and siCON1 on day 0. Squares, Neuro2A-Luc (dt = 0.8 d); diamonds, LNCaP-Luc (dt = 1.4 d); triangles, HeLa-Luc (dt = 1.6 d); circles, CCD-1074Sk-Luc (nondividing). (B) Luciferase knockdown after siRNA transfection predicted by the mathematical model using the baseline in vitro parameters given in Table 2 with the initial number of dividing and nondividing cells equal to 5 × 104 and 1.5 × 105, respectively, a transfection time of 5 h, and a transfection efficiency of 90%.
Figure 4
Figure 4
Kinetics of luciferase knockdown by siRNA in Neuro2A-Luc subcutaneous tumors in A/J mice. (A) Experimental and predicted results for luciferase knockdown after three consecutive LPTV injections on days 6, 7 and 8 of transferrin-targeted CDP-Im polyplexes containing 50 µg siRNA per 20 g mouse. Experimental data points are shown for a mouse receiving siCON1 (squares) and a mouse receiving siGL3 (circles). Solid lines represent the predicted luciferase signal with siRNA treatment and dashed lines represent the predicted luciferase signal in the absence of siRNA treatment. (B) Normalization of the observed luciferase signal in the siGL3-treated mouse to the predicted luciferase signal in the absence of treatment. Circles indicate the normalized experimental data points, while the solid line represents the response predicted by the mathematical model using the baseline in vivo parameters given in Table 2 and assuming that 50% of the total cells are reached with each dose.
Figure 5
Figure 5
Kinetics of luciferase knockdown by siRNA in nondividing hepatocytes in BALB/c mice. Experimental and predicted results are shown for luciferase knockdown after hydrodynamic tail-vein co-injection of 5 µg pApoEHCRLuc and 50 µg siRNA per 20 g mouse on day 0. Circles represent the ratio of the average luciferase signal intensity from three mice receiving plasmid + siRNA to the luciferase signal intensity from three mice receiving plasmid alone. The predicted luciferase knockdown, given by the solid line, was calculated using the baseline in vivo parameters given in Table 2 with the following modifications to account for hydrodynamic injection of naked siRNA without a delivery vehicle: eliminate steps involving the complexes (kescendvec, kunpackend, kunpackcyt), modify uptake and intracellular trafficking to match observed kinetics (partition = 1 × 10−2, ktransblood = 1, kint = 1 ×10−3 h−1, kescendna = 1 × 10−2 h−1, kdegendna = 5 × 10−3 h−1), and modify extracellular volume (Ve = 1.5 × 10−5 L). The kescendna and kdegendna may no longer represent endosomal processes as hydrodynamically injected naked siRNA may be internalized through different vesicles or partitioned into a separate intracellular compartment (e.g. nucleus) that exhibits different degradation and release kinetics than in standard or receptor-mediated endocytosis of siRNA-containing complexes. The total number of hepatocytes was chosen to be 5 × 107, on the same order of magnitude as the number of hepatocytes in a mouse liver (40,41).
Figure 6
Figure 6
Effect of intracellular siRNA half-life on the duration of siRNA-mediated gene silencing in vitro. Curves represent model predictions for luciferase knockdown after transfection with 100 nM siRNA against luciferase on day 0 with a cell doubling time of 1 day (kgrowth = 0.0.029 h−1) and intracellular siRNA half-lives of 24, 48 and 72 h (kdeginna = 0.029, 0.014 and 0.01 h−1). The initial number of cells was 5 × 104, transfection time was 5 h, transfection efficiency was 90%, and all other parameters were kept at their baseline in vitro values given in Table 2.
Figure 7
Figure 7
Effect of siRNA dose frequency on the duration of luciferase knockdown by siRNA in nondividing cells. (A) Experimental results using Oligofectamine to deliver siRNA to luciferase-expressing nondividing fibroblasts in vitro. Data points represent the ratio of the average luciferase signal intensity from triplicate wells receiving siGL3 and siCON1. To facilitate comparison of the knockdown kinetics, the data points are normalized such that all three curves exhibit the same magnitude of knockdown for the first four days since all three received the same treatment over this period. This normalization permits comparison of the kinetics of gene silencing observed with different treatments even though the absolute magnitude of the knockdown varied slightly in each experiment. Squares, 100 nM (day 0); diamonds, 100 nM (day 0) + 10 nM (day 4); triangles, 100 nM (day 0) + 100 nM (day 4). (B) Luciferase knockdown after siRNA transfection predicted by the mathematical model using the baseline in vitro parameters given in Table 2 with the number of cells equal to 1.5 × 105, a transfection time of 5 h, and a transfection efficiency of 90%.
Figure 8
Figure 8
Effect of cell doubling time and target protein half-life on the ability to maintain persistent gene silencing. All plots represent predicted mRNA (dashed lines) and protein (solid lines) knockdown in transfected cells using the baseline in vitro parameters given in Table 2, a transfection time of 5 h, and an initial number of dividing and nondividing cells equal to 5 × 104 and 1.5 × 105, respectively. (A) Dose of 100 nM siRNA every 3 days with a target protein half-life of 2 h (kdegprot = 0.35 h−1) in cells with a doubling time of 1 day (kgrowth = 0.029 h−1). (B) Dose of 100 nM siRNA every 3 days with a target protein half-life of 48 h (kdegprot = 0.014 h−1) in cells with a doubling time of 1 day (kgrowth = 0.029 h−1). (C) Dose of 100 nM siRNA every 7 days with a target protein half-life of 2 h (kdegprot = 0.35 h−1) in nondividing cells. (D) Dose of 100 nM siRNA every 7 days with a target protein half-life of 48 h (kdegprot =0.014 h−1) in nondividing cells. (E) Effect of variations in cell doubling time and target protein half-life on the ability to maintain a target protein level below a threshold of 50% its pre-treatment value over the 25-day period. I, 100 nM (day 0); II, 100 nM (days 0, 7, 14); III, 100 nM (days 0, 3, 7, 10, 14, 17, 21, 24). Surface vertices represent the fraction of the total time during which the relative protein level is below the 50% threshold.
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