Impact of Phosphorothioate Chirality on Double‐Stranded siRNAs: A Systematic Evaluation of Stereopure siRNA Designs

Abstract

To mL eedom, Mark Foster,a nd Curt W. Bradshaw [a] Oligonucleotides are important therapeutic approaches, as evidenced by recent clinicals uccesses with antisense oligonucleotides (ASOs)a nd double-stranded short interfering RNAs (siRNAs). Phosphorothioate (PS) modifications are as tandard feature in the current generation of oligonucleotide therapeutics, but generatei someric mixtures, leadingt o2 n isomers. All currently marketed therapeutic oligonucleotides (ASOs and siRNAs)a re complex isomeric mixtures. Recent chemical methodologiesf or stereopure PS insertions have resulted in preliminary rules for ASOs, with multiple stereopure ASOs moving into clinicald evelopment. Although siRNAs have comparatively fewer PSs, the field has yet to embrace the idea of stereopure siRNAs.H erein, it has been investigated whether the individual isomersc ontributee qually to the in vivo activity of ar epresentative siRNA. The results of as ystematic evaluation of stereopure PS incorporation into antithrombin-3 (AT3) siRNA are reported and demonstrate that individualP Sisomersd ramatically affect in vivo activity.Astandard siRNA design with six PS insertions was investigated and it was found that only about 10 %o ft he 64 possible isomersw ere as efficacious as the stereorandomc ontrol.B ased on this data, it can be concluded that G1R stereochemistry is critical, G2R is important,G 21S is preferable, and G22 and P1/P2 tolerate both isomers. Surprisingly,t he disproportionate loss of efficacy for most isomers does not translate into significant gain for the productive isomers, and thus, warrants further mechanistic studies.Since the discovery by Fire and Mello in 1998 that doublestranded short interfering RNA (siRNA) can cleave mRNA and inhibit protein translation, [1] this new class of therapeutics has moved towards clinical utility.T he advent of double-stranded siRNAs as am odality to harnessanatural catalytic pathway, called RNA interference (RNAi), excited scientific and business communities alike because of its implications in therapeutics, particularly for targets difficult to drug with small-molecules and proteins. [2] Interesti ns iRNAs has had periodic boom and bust cycles following the recognition of and solutions for technical challenges. One of the significant challenges is delivery of these large, charged siRNA molecules across the cell membrane.B oth lipid nanoparticle and ligand-based approaches are clinically validated, with ar ecent approvalo ft he first RNAi therapeutic, Patisiran, al ipid complex from Alnylam, and nu-merousG alNAc-targeteds iRNAs in clinical trials. [3] Naturally occurring siRNA molecules have two complementary strands (19b ase pairs), with an additional two unpaired bases at the 3'-ends, so-called 21/21. Aw ide variety of variations weree xploredi ne arly optimization, includings ignificant modifications in size (19-to 27-mers) and shape (Dicer-substrate siRNA, asymmetric and blunt-ended designs). [4] In addition, siRNA components, such as sugars,n ucleobases, and the phosphate backbon...

Chirality matters: stereo-defined phosphorothioate linkages at the termini of small interfering RNAs improve pharmacology in vivo

“…In line with previous reports ( 36 , 51 ), including our patent disclosure ( https://patents.google.com/patent/WO2019126651A1/en ), this account provides further demonstration of how chiral PS linkages at the termini of an siRNA can be exploited to optimize RISC loading and metabolic stability. In particular, the findings of the study suggest that careful selection of PS stereochemistry at defined positions of the siRNA antisense strand results in benefits stemming from the different properties of R p and S p PS isomers.…”

Identification of a Tricyclic PIII Chiral Auxiliary for Solid‐Supported Synthesis of Stereopure Phosphorothioate‐Containing Oligonucleotides

“…Compared with ASOs, siRNAs use fewer (<10) PS at the guide‐ and passenger‐strand termini. We recently discovered about 10 % of the isomers of current siRNA design disproportionately contributed to the activity, in which some of the individual isomers are as active or slightly more active than that of a mixture of about 64 isomers . Our interest in stereopure PS‐containing siRNAs prompted us to develop a practical method and we report our findings herein.…”

“…Finally, we identified a new DHI auxiliary that provided excellent diastereoselectivity and was easier to synthesize. Our recent findings of the impact of stereopure PSs on siRNA efficacy, combined with progress in novel chiral auxiliary based stereopure PS insertion methods, are likely to pave the way for the application of stereopure siRNAs as therapeutics.…”

Phosphorothioate Substitutions in RNA Structure Studied by Molecular Dynamics Simulations, QM/MM Calculations and NMR Experiments

“…28,36 Remaining imino resonances could be assigned with imino-imino cross-peaks of 2D-1 H, 1 H-NOESY spectra. 2D-1 H, 1 H-NOESY, 2D-1 H, 15…”

“…12 Separate synthesis of the two enantiomers or purification of their mixtures are non-trivial issues and subject of ongoing experimental research. [13][14][15] On the other hand, the PT enantiomer can be unambiguously selected in theoretical studies. Despite their significant biological effects on RNA metabolism, the structural and dynamical impacts of PT modifications are poorly understood.…”