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. 2005 Oct;89(4):2286-95.
doi: 10.1529/biophysj.104.054080. Epub 2005 Jul 29.

Molecular and cellular barriers limiting the effectiveness of antisense oligonucleotides

Charles M Roth  1
Affiliations

Molecular and cellular barriers limiting the effectiveness of antisense oligonucleotides

Charles M Roth. Biophys J. 2005 Oct.

Abstract

Antisense oligonucleotides present a powerful means to inhibit expression of specific genes, but their effectiveness is limited by factors including cellular delivery, biochemical attack, and poor binding to target. We have developed a systems model of the processes required for an antisense oligonucleotide to enter, gain access to its target mRNA, and exert activity in a cell. The model accurately mimics observed trends in antisense effectiveness with the stability of the oligonucleotide backbone and with the affinity/kinetics of binding to the mRNA over the time course of inhibition. By varying the model parameters within the physically realizable range, we note that the major molecular and cellular barriers to antisense effectiveness are intracellular trafficking, oligonucleotide-mRNA binding rate, and nuclease degradation of oligonucleotides, with a weaker dependence on total cellular uptake than might be expected. Furthermore, the model may serve as a predictive tool to design and test strategies for the cellular use of antisense oligonucleotides. The use of integrated mathematical modeling can play a significant role in the development of antisense and related technologies.

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Figures

FIGURE 1
FIGURE 1
Schematic diagram of major interactions in the model for antisense delivery and activity. AS ONs (shown in red throughout) in the extracellular environment (Ab) bind to cellular surface molecules (R) and are internalized by endocytosis. The oligonucleotides that are internalized (Ai) must escape the endosome to enter the cytoplasm (Ac) before routing and degradation in lysosomes (X). Once in the cytoplasm, AS ONs can find their complementary target mRNA (M) and hybridize to form a duplex (D). The duplex is incapable of translation, and the mRNA portion may be degraded by Rnase H. Hybridization of cytoplasmic oligonucleotides to nontarget mRNA (N) is also possible, and these duplexes would also serve as substrates for Rnase H. Definition of all variables can be found in the main text.
FIGURE 2
FIGURE 2
Effect of model parameters on cytoplasmic concentrations. (A) Effect of bulk oligonucleotide on cytoplasmic concentration, Ac. Curves correspond to bulk concentrations (Ab) of {200, 400, 600, 800, and 1000} nM. (B) Effect of endosomal escape rate on Ac. Curves correspond to escape rate constants (kt) of {0.001, 0.003, 0.010, and 0.030} min−1.
FIGURE 3
FIGURE 3
Interplay of uptake and activity dynamics. (A) Predicted difference in cytoplasmic concentration of a fast binding (ka = 10.0 μM−1 min−1) versus nonbinding (ka = 0) oligonucleotide. Base case parameters were used in the model except for a cytoplasmic degradation rate of 0.003 min−1. (B) Experimental results for uptake of fluorescently labeled oligonucleotides that are complementary (A-ODN157) or noncomplementary (A-ODNcontrol) as a function of time after delivery to CHO-pd1EGFP cells, as determined by flow cytometry. (C) Predicted dynamics of antisense activity for the same parameter set as in A. (D) Experimentally determined geometric mean pd1EGFP fluorescence as determined by flow cytometry for treatment of the same samples analyzed in B for the complementary oligonucleotide A-ODN157. Experimental values shown are mean ± SE, with the error bars in D being mostly smaller than the symbols.
FIGURE 4
FIGURE 4
Effect of random variation in model parameters on predictions. The antisense activity (change in target mRNA levels) was simulated for the base case parameters and is shown in the solid line. The values of all model parameters were each varied randomly within 10% (dotted lines) or 20% (dashed lines), and the range of mRNA dynamics resulting from 100 runs are shown.
FIGURE 5
FIGURE 5
Effect of bulk concentration and trafficking on antisense inhibition. The minimum target mRNA levels, normalized to the initial values, as a function of (A) bulk oligonucleotide concentration and (B) endosomal escape rate. In both cases, the minima occurred at times between 210 and 240 min.
FIGURE 6
FIGURE 6
Interplay of hybridization rate and stability on antisense activity. (A) Predicted mRNA dynamics as a function of hybridization rate for (A) unstable (δm = 0.03 min−1), (B) moderately stable (δm = 0.003 min−1), and (C) very stable (δm = 0.0003 min−1) oligonucleotides. In each case (A–C), the hybridization rate constants are ka = {0.1, 1.0, and 10.0} μM−1 min−1. (D) Experimentally observed dynamics, measured using real-time polymerase chain reaction of the rat gp130 mRNA in H35 cells treated with PS (moderate stability) oligonucleotides selected for fast binding and slow binding, respectively.
FIGURE 7
FIGURE 7
Effect of Rnase H degradation on antisense activity. Minimum mRNA levels, normalized to the initial values, as a function of the first-order rate constant for Rnase H-mediated target degradation, for moderate oligonucleotide-mRNA binding (ka = 1.0 μM−1 min−1), and for more rapid oligonucleotide-mRNA binding (ka = 10.0 μM−1 min−1).
FIGURE 8
FIGURE 8
Effect of mRNA decay on antisense inhibition. (A) The mRNA decay rate (δm) was varied with the initial mRNA level held constant at 500 copies per cell, i.e., the mRNA synthesis rate (σm) was varied correspondingly. Individual curves correspond to mRNA decay rates of {0.001, 0.003, 0.01, 0.03, and 0.1} min−1. (B) The mRNA decay rate was varied with the mRNA synthesis rate held constant, thus resulting in a decrease in initial mRNA level with increasing decay rate. Individual curves correspond to mRNA decay rates of {0.003, 0.075, 0.03, 0.075, 0.3, and 0.75} min−1.
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