Characterization of molecular and cellular events limiting the effectiveness of antisense oligonucleotides

Antisense oligonucleotides (AS ONs) are a powerful means to inhibit the expression of specific genes, but their effectiveness is limited by factors including cellular delivery, biochemical attack, and poor binding to target. We are developing rational approaches to improve effectiveness through high affinity binding. We developed a molecular thermodynamic model based on ON and mRNA folding that predicts accurately the affinity of ON-mRNA binding, and we have found that it successfully selects those ONs that are most successful in cell culture. We have experimentally measured both thermodynamic and kinetic binding events in solution and found strong correlation between association kinetics and binding affinity. Binding association rates as a function of temperature have provided evidence for our hypothesis that RNA restructuring is the limiting step in ON binding. We are currently integrating these results with additional measurements on delivery and ribonuclease H cleavage of RNA in DNA-RNA duplexes to form a whole cell model that can serve as the basis for optimization of ON chemistry, dosing and timing. Taken together, these tools may help accelerate the development of this promising technology.