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.

Keywords: DNA; RNA; RNase H; antisense; mechanisms; oligonucleotide.

FIG. 1.

Rates of steps in RNase H1 activating ASOs activity [11]. (A) After transfection, about 60 min is required for intracellular distribution of ASOs. In both the nucleus and cytoplasm, about 20 min are required to screen nucleic acid sequence and bind to the cognate site. Approximately 40 min are then required to recruit RNase H1 and achieve measurable RNA target reduction. (B) The transcription and splicing rates for the wild-type SOD1 construct and a mutant (187) with substantially reduced splicing. (C) An effective RNase H1 activating ASO approximately doubles the intrinsic rate of cellular RNA degradation. (D) The concentration of RNAse H1 is rate limiting. Over-expression of Escherichia coli RNase H1 again doubles the rate reduction induced by an effective RNase H1 ASO. ASO, antisense oligonucleotide.

FIG. 1.

Rates of steps in RNase H1 activating ASOs activity [11]. (A) After transfection, about 60 min is required for intracellular distribution of ASOs. In both the nucleus and cytoplasm, about 20 min are required to screen nucleic acid sequence and bind to the cognate site. Approximately 40 min are then required to recruit RNase H1 and achieve measurable RNA target reduction. (B) The transcription and splicing rates for the wild-type SOD1 construct and a mutant (187) with substantially reduced splicing. (C) An effective RNase H1 activating ASO approximately doubles the intrinsic rate of cellular RNA degradation. (D) The concentration of RNAse H1 is rate limiting. Over-expression of Escherichia coli RNase H1 again doubles the rate reduction induced by an effective RNase H1 ASO. ASO, antisense oligonucleotide.

FIG. 2.

Subcellular distribution of PS ASOs and cleavage of target RNA by RNase H1. PS ASO distribute within the cell via interactions with specific proteins. Once the ASO has bound to its cognate site in the target RNA, RNase H1 (and P32) is recruited to degrade the target RNA. Post-RNase H1 cleavage and the fragments are processed by normal cellular RNA degradation pathways. PS, phosphorothioate.