Stereochemical bias introduced during RNA synthesis modulates the activity of phosphorothioate siRNAs

Jahns, Hartmut; Roos, Martina; Imig, Jochen; Baumann, Fabienne; Wang, Yuluan; Gilmour, Ryan; Hall, Jonathan

doi:10.1038/ncomms7317

Cited by 79 publications

(55 citation statements)

References 48 publications

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“…One underappreciated outcome of PS insertions is the introduction of stereoisomers relative to the non‐stereomeric PO backbone (Figure ). Standard oligonucleotide synthesis lacks stereocontrol, but the issue was ignored primarily due to the success of PS‐containing oligonucleotides in the clinic and lack of practical stereopure PS synthetic methodology. Stereopure PScontaining oligonucleotides became a reality with the development of oxazaphospholidine methods .…”

Section: Figurementioning

confidence: 99%

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

et al. 2020

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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...

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Section: Figurementioning

confidence: 99%

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

et al. 2020

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“…reported that the PS‐siRNAs composed of oligoribonucleotides (ORNs) with a higher fraction of Rp centres showed greater resistance to nucleases in serum and were more effective inhibitors in cells than their Sp counterparts. Introduction of phosphorothioate (PS) modifications in the place of phosphodiester linkages of siRNA exhibited improved potency when tested in HeLa cells by luciferase assay …”

Section: Phosphate Backbone Modificationsmentioning

confidence: 99%

Therapeutic potential of chemically modified siRNA: Recent trends

et al. 2017

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Small Interfering RNAs (siRNAs) are one of the valuable tools to investigate the functions of genes, and are also used for gene silencing. It has a wide scope in drug discovery through in vivo target validation. siRNA therapeutics are not optimal drug-like molecules due to poor bioavailability, immunogenic and off-target effects. In order to overcome the challenges associated with siRNA therapeutics, identification of appropriate chemical modifications that improves the stability, specificity and potency of siRNA is essential. This review focuses on the various chemical modifications and their implications in siRNA therapy.

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“…: 6655.3; ref. 40). Chemicals for ORN synthesis were from Aldrich and TCI (Sigma-Aldrich Chemie GmbH, D-89555 Steinheim).…”

Section: Synthesis Of Sirna For In Vivo Studiesmentioning

confidence: 99%

“…This previously characterized siRNA (siLin28B-2) was selected for its high knockdown efficiency (40). Transfection of siLin28B-2 reduced Lin28B mRNA and concomitantly increased let-7b expression in DU145 cells ( Supplementary Fig.…”

Section: Targeting Of Lin28 In Prostate Cancer Cells Blocks Csc Self-mentioning

confidence: 99%

Activation of the Lin28/let-7 Axis by Loss of ESE3/EHF Promotes a Tumorigenic and Stem-like Phenotype in Prostate Cancer

et al. 2016

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Although cancer stem-like cells (CSC) are thought to be the most tumorigenic, metastatic, and therapy-resistant cell subpopulation within human tumors, current therapies target bulk tumor cells while tending to spare CSC. In seeking to understand mechanisms needed to acquire and maintain a CSC phenotype in prostate cancer, we investigated connections between the ETS transcription factor ESE3/EHF, the Lin28/ let-7 microRNA axis, and the CSC subpopulation in this malignancy. In normal cells, we found that ESE3/EHF bound and repressed promoters for the Lin28A and Lin28B genes while activating transcription and maturation of the let-7 microRNAs. In cancer cells, reduced expression of ESE3/EHF upregulated Lin28A and Lin28B and downregulated the let-7 microRNAs. Notably, we found that deregulation of the Lin28/let-7 axis with reduced production of let-7 microRNAs was critical for cell transformation and expansion of prostate CSC. Moreover, targeting Lin28A/Lin28B in cell lines and tumor xenografts mimicked the effects of ESE3/EHF and restrained tumor-initiating and self-renewal properties of prostate CSC both in vitro and in vivo. These results establish that tight control by ESE3/EHF over the Lin28/let-7 axis is a critical barrier to malignant transformation, and they also suggest new strategies to antagonize CSC in human prostate cancer for therapeutic purposes. Cancer Res; 76(12); 3629-43. Ó2016 AACR.

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Stereochemical bias introduced during RNA synthesis modulates the activity of phosphorothioate siRNAs

Cited by 79 publications

References 48 publications

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

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

Therapeutic potential of chemically modified siRNA: Recent trends

Activation of the Lin28/let-7 Axis by Loss of ESE3/EHF Promotes a Tumorigenic and Stem-like Phenotype in Prostate Cancer

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