A Kinetic Model Explains Why Shorter and Less Affine Enzyme-recruiting Oligonucleotides Can Be More Potent

Abstract

Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.

Figure 1

Schematic of the modeled reactions. Initially the oligonucleotide (O) binds to the target (T) and forms the OT complex, which recruits the enzyme (E). Within the OTE complex, the target is cleaved (C) to form OCE. The enzyme and then the oligonucleotide dissociates from the cleaved target to enter a new round of catalysis. The target has a constant production rate denoted by ν_prod, and a basal oligonucleotide- and RNase H-independent degradation rate, . Here, denotes completely degraded target. The dissociation rate of enzyme from OT and OC is assumed to be the same.

Figure 2

Model solutions and identification of an optimal binding affinity. (a,b) Time-resolved simulation of the relative concentrations of key species in the reaction scheme from Figure 1. Oligonucleotide (O) is added at time t = 0 minute at a concentration of (a) 0.1 and (b) 100 nmol/l. (c) The relative total target concentration (T_rel) as a function of total oligonucleotide concentration at typical parameter settings (Table 1). Dashed lines indicate efficacy (horizontal) and half-maximal effect concentration (EC₅₀) (vertical). Arrows indicate the total oligonucleotide concentrations used in (a) and (b). (d) The EC₅₀ as a function of the dissociation constant for the OT complex. A low dissociation constant between the oligonucleotide and the target (K_dOT) corresponds to a high binding affinity. Dashed line: no coupling between and . Solid line: . (e–g) Experimental knockdown as a function of calculated ΔG°: (e) for 21 oligonucleotides at 2 nmol/l targeted against the luciferase firefly gene, (f) for 14 oligonucleotides at 3 nmol/l targeted against the glucocorticoid receptor, and (g) for 23 oligonucleotides at 1 nmol/l targeted against apolipoprotein B (APOB), (h) 4 oligonucleotides at 0.06 μmol/l and 1.5 μmol/l against PCSK9. Legend indicates oligonucleotide lengths. Target messenger RNA concentrations are measured by (e) luciferase assay and (f,g,h) quantitative reverse transcriptase–polymerase chain reaction. Dots are experimental data and gray lines are a least squares fit to a second-order polynomial with P values and vertex for the fit in each of the panels e–h.

Figure 3

Schematic drawing of target cleavage illustrating the rationale for introducing the coupling . Upon enzyme (E, RNase H) binding to the OT complex the target is cleaved at a rate . After cleavage, the right and left parts of the cleaved target will dissociate from the oligonucleotide at (faster) rates k_right and k_left, respectively.

Figure 4

The optimal model-predicted affinity is dependent on the physiological parameters. The half-maximal effect concentration (EC₅₀) is plotted against the binding affinity, quantified by dissociation constant between the oligonucleotide and the target (K_dOT), while varying (a) the total RNase H concentration, E_t, (b) the coupling constant, α, (c) the target production, ν_prod, (d) the target degradation, , (e) the dissociation constant for the OTE complex, K_dOTE, and (f) the rate of target cleavage, .