Single-Stranded Phosphorothioated Regions Enhance Cellular Uptake of Cholesterol-Conjugated siRNA but Not Silencing Efficacy

Abstract

Small interfering RNAs (siRNAs) have potential to silence virtually any disease-causing gene but require chemical modifications for delivery to the tissue and cell of interest. Previously, we demonstrated that asymmetric, phosphorothioate (PS)-modified, chemically stabilized, cholesterol-conjugated siRNAs, called hsiRNAs, support rapid cellular uptake and efficient mRNA silencing both in cultured cells and in vivo . Here, we systematically evaluated the impact of number, structure, and sequence context of PS-modified backbones on cellular uptake and RNAi-mediated silencing efficacy. We find that PS enhances cellular internalization in a sequence-dependent manner but only when present in a single-stranded but not double-stranded region. Furthermore, the observed increase in cellular internalization did not correlate with functional silencing improvement, indicating that PS-mediated uptake may drive compounds to non-productive sinks. Thus, the primary contributing factor of PS modifications to functional efficacy is likely stabilization rather than enhanced cellular uptake. A better understanding of the relative impact of different chemistries on productive versus non-productive uptake will assist in improved design of therapeutic RNAs.

Tuning G-Quadruplex Nanostructures with Lipids. Towards Designing Hybrid Scaffolds for Oligonucleotide Delivery

“…Figure 11 shows the luciferase concentration obtained after treatment of HeLa cells with these oligonucleotides (60 nM) after 24, 48 and 72 h. This result shows a clear inhibitory advantage for the G-rich oligonucleotide. This increased efficacy might result from a positive contribution of the G-rich structure in the endosomal escape [61] and/or an increased efficacy of the oligonucleotide in the binding and subsequent RNase H degradation of target luciferase mRNA [62]. Next, we evaluated the long-term silencing properties of these two oligonucleotides (25 and 26) and Luc oligonucleotide without using a transfecting reagent.…”

Antiphospholipid Syndrome and Pregnancy-Diagnosis, Complications and Management: An Overview

“…Complications of anticoagulant therapy in pregnancy include embryopathy (nasal hypoplasia, spotted epiphyses), CNS abnormalities (Dandy-Walker syndrome, visual atrophy), fetal bleeding, hemorrhagic manifestations, skin allergies, thrombocytopenia and osteoporosis [60][61][62][63][64][65][66][67][68][69][70][71][72].…”

“…Treatment should be applied only when the risk of complications is considered to be higher and after a thorough discussion with the pregnant woman. The prognostic factors of poor outcome are the title of anticardiolipin antibodies and the obstetric history [60][61][62][63][64][65][66][67][68][69][70][71][72].…”

“…Until now, there are no final conclusions regarding the efficacy of the above therapy as monotherapy. Hydroxychloroquine inhibits aPL-B2GPI complexes on phospholipid surfaces, annexin A5, TF tissue factor, TLPs Toll-like receptors, statins such as pravastatin inhibition of Nuclear factor kappa B (NF-κB) is protein transcription factor, Anti-CD20 monoclonal antibodies (mAbs) resistant APS and aspirin are recommended to prevent and treat preeclampsia [60][61][62][63][64][65][66][67][68][69][70][71][72].…”

Cowpea Chlorotic Mottle Virus‐Like Particles as Potential Platform for Antisense Oligonucleotide Delivery in Posterior Segment Ocular Diseases

“…[ 16 ] As in this case, therapeutic nucleic acids (TNAs) under preclinical and clinical investigation are generally chemically modified in order to increase their stability (e.g., against endonucleases and exonucleases) and efficacy. [ 23–29 ] Most commonly used modifications include phosphorothioate oligos, 2′‐ O ‐methyl‐modified oligos and locked nucleic acids (LNAs). For instance, in 1998 the first phosphorothioate antisense oligonucleotide Fomiversen (Vitravene) was approved for the treatment of cytomegalovirus retinitis and in 2004 Pegaptanib (Macugen) has been approved for the treatment of wet AMD.…”