Molecular Mechanisms of Antisense Oligonucleotides

Abstract

In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry and began work to understand how oligonucleotides behave in biological systems. Since 1989, my research has focused primarily on this topic, although I have been involved in most areas of research in antisense technology. I believe that the art of excellent science is to frame large important questions that are perhaps not immediately answerable with existing knowledge and methods, and then conceive a long-term (multiyear) research strategy that begins by answering the most pressing answerable questions on the path to the long-term goals. Then, a step-by-step research pathway that will address the strategic questions posed must be implemented, adjusting the plan as new things are learned. This is the approach we have taken at Ionis. Obviously, to create antisense technology, we have had to address a wide array of strategic questions, for example, the medicinal chemistry of oligonucleotides, manufacturing and analytical methods, pharmacokinetics and toxicology, as well as questions about the molecular pharmacology of antisense oligonucleotides (ASOs). Each of these endeavors has consumed nearly three decades of scientific effort, is still very much a work-in-progress, and has resulted in hundreds of publications. As a recipient of the Lifetime Achievement Award 2016 granted by the Oligonucleotide Therapeutic Society, in this note, my goal is to summarize the contributions of my group to the efforts to understand the molecular mechanisms of ASOs.

Observing an Antisense Drug Complex in Intact Human Cells by in‐Cell NMR Spectroscopy

“…On the basis of these results we hypothesized that the ASO forms large complexes, thus leading to signal broadening and signal loss in the case of solution‐state NMR. This is consistent with the literature, in which ASO is expected to exist in complexes with the target mRNA and RNaseH1 (active complex), in addition to interacting with trafficking proteins or chaperones …”

Versatile Method for the Site‐Specific Modification of DNA with Boron Clusters: Anti‐Epidermal Growth Factor Receptor (EGFR) Antisense Oligonucleotide Case

“…By contrast, duplexes of 13.2 ‐type oligomers containing U B ‐type modifications at positions‐7, ‐11, or both exhibited similar thermal stability (Δ T m =7.5 °C for DNA/DNA ( 13.2 c8 vs. 13.2 b6 ) and 4.0 °C for DNA/RNA duplexes ( 13.2 c8 vs. 13.2 b6 )). The thermodynamic stability of the boron cluster‐DNA/RNA duplexes with U B ‐type‐modified units would be advantageous for the application of these modified ASOs for the recognition of their complementary mRNA sequences and duplex formation, a prerequisite for gene expression regulation by the antisense approach …”

Targeting RNA: A Transformative Therapeutic Strategy

“…In principle, oligonucleotides can be rationally designed against virtually any genetic target . Their unique mechanism of action differentiates this class of therapeutics from small molecules and protein therapeutics ( Table ) . Oligonucleotides bind to their cognate RNA target by Watson‐Crick hybridization with high selectivity and affinity.…”

“…The mechanism of action, chemical evolution, and intracellular delivery of oligonucleotide therapies are only briefly reviewed to provide a background for this class of therapies. Reviews specific in these areas have been published elsewhere and the readers are encouraged to review them …”