Session Summary
Keynote presentation by Stan Crooke, Executive Chairman of Ionis Pharmaceuticals
Summarized by biotech researcher and writer Catarina Carrao
Dr. Stan Crooke, Executive Chairman of Ionis Pharmaceuticals, started by emphasizing that antisense technology is beginning to deliver on the broad promise more than three decades ago. Twelve RNA Targeted drugs (RTDs) including 8 single strand antisense drugs (ASOs) and 2 double strand ASOs (siRNAs) have now been approved for commercial use and the ASOs in Phase 2/3 trials are innovative, delivered by multiple routes of administration and focused on both rare and common diseases. In fact, two ASOs are in cardiovascular outcome studies and several others in very large trials, showing how this technology is disseminating.
Today there are two structural classes that are used broadly – single-stranded (ssASO) and double-stranded (ds ASO) RTDs - where most, some or all of the inner nucleotide linkages are substituted with phosphorothioate (PS), which has revealed itself very useful because it enhances and optimizes protein binding.
The general principles of PS-ASO protein interactions reveal that proteins determine the fate of PS-ASOs; that is, if a PS-ASO is present in a biological site, a protein or protein complex is responsible for its location. In the other way, also PS-ASOs alter the fates of many proteins, where multiple protein aggregates are formed at various concentrations of PS-ASOs Welp,in cells with some being RNPs normally found in cells, and other uniquely formed only in the presence of PS-ASOs. Diverse types of proteins interact with PS-ASOs; and although nucleic acid binding proteins play important roles in the binding of PS-ASOs, many other domains are also involved.
Dr. Crooke explained that phosphorothioate content is required for binding to most proteins. In ASOs with both PO and PS linkages, the placement of the PS moieties can influence interactions with some proteins; however, no “consensus” PS pattern seems to be apparent. Many proteins demonstrate a strong preference to bind the 5’ terminus of PS-ASOs; and the most likely explanation, according to Dr. Crooke, is that the proteins sense the partial helicity of PS-ASOs. Very importantly, many proteins discriminate between 2’ modifications, and can distinguish between PS-ASOs of different sequences. Also, ionic, hydrophobic and base aminoacid stacking interactions are critical; with water of hydration and counterions present, but with no discernible effect on PS-ASO protein interactions. In relation to membrane phospholipids, they may affect PS-ASO protein interactions; but, so far, no direct interaction has been identified between PS-ASOs and lipid components of the membrane.
Relative to PS-ASO-induced conformational change and protein-protein interactions, it is now known that PS-ASO can cause protein conformational change, which is affected by PS-ASO binding affinity. PS-ASO can also form different ASO-protein structures in cells, including paraspeckles, with liquid-like structures. Also, PS-ASO binding can alter protein localization and phase separation, which allows ASO internalization and trafficking. According to Dr. Crooke, the kinetics of PS-ASO interactions in cells are much slower than small molecule drugs, given the example of the RNase H1 PS-ASO kinetics, which is well characterized. Also, at this point, many factors are known to affect ASO activity, like RNA structure, protein binding, splicing rate, number of cognate binding sites, subcellular localization, position of binding site, RNA degradation/translation rate, target protein half-life and RNase H1 level. Transcription rate and copy number doesn’t seem to affect ASO activity in Dr. Crooke’s words.
There are mechanisms by which PS-ASOs can induce toxicity (excluding on-target mediated toxicity); and, the most important of all is the nucleolar mis-localization of cellular proteins by toxic cEt ASOs mediated by RNase H1. The toxic ASO induces cloudy nucleolar structures that are liquid-like; and, in cells that survived toxic PS-ASO, different nucleolar structures can form at a later time.
Antisense technology is beginning to deliver value to patients, with the evolution of medical chemistry and our understanding of the molecular mechanisms of distribution, pharmacodynamics and toxicity supporting the creation of a new, even more exciting future. New generation PS-ASOs will likely have several chemical decorations, for target delivery and maximized therapeutics index. For Dr. Crooke, this is the realization of the ‘designer drug’ dream.