Phosphorothioate-Stimulated Cellular Uptake of siRNA: A Cell Culture Model for Mechanistic Studies

Abstract

The phosphorothioate(PS)-stimulated cellular uptake of naked short interfering RNA (siRNA) into mammalian cells indicates a promising new mechanistic strategy because it makes use of a caveosomal, rather than an endosomal pathway, which is used by the majority of known delivery systems. This PS-stimulated mode delivers large amounts of siRNA primarily into the perinuclear space which is related to measurable though moderate target suppression. The observed limited efficacy seems to be related to intracellular trapping of siRNA. Here, we studied the intracellular localisation of siRNA and Argonaute 2 (Ago2), the major component of the RNA interference (RNAi) machinery, by density gradient centrifugation and fluorescence microscopy after PS-stimulated delivery or transfection with Lipofectamine 2000. The two cell lines ECV-304 and SKRC-35 both take up siRNA in the PS-stimulated mode but only ECV-304 shows RNAi, i.e. siRNA-mediated suppression of lamin A/C expression, whereas SKRC-35 does not. This lack of RNAi in the latter cell line seems to be due to a block of an intracellular siRNA translocation process. This study provides strong evidence for the view that co-localisation of siRNA and Ago2 in the vicinity of the rough endoplasmic reticulum (rER) in ECV-304 cells is related to target inhibition, whereas density gradient fractionation of cell organelles shows a lack of co-localisation in SKRC-35 cells in which RNAi does not occur after the PS-mediated delivery. In summary, we propose to exploit this dual cell system to identify important steps of intracellular trafficking of siRNA after PS mediated delivery that are crucial for its biological activity and which seem to be of general importance for the understanding of the intracellular trafficking and release of siRNA.

Selection, Optimization, and Pharmacokinetic Properties of a Novel, Potent Antiviral Locked Nucleic Acid-Based Antisense Oligomer Targeting Hepatitis C Virus Internal Ribosome Entry Site

“…Assuming a 1:1 molar stoichiometry in the antisense mechanism of action, one would theoretically expect antisense modalities to be efficacious in the femtomolar range. Our data, however, reinforce observations by others that collectively point to a rate-limiting step(s), including cellular uptake, intracellular transport, and differential cellular compartmentalization, of HCV and oligonucleotide therapeutics (3,8,10,25).…”

The Biology of CNAPS

“…Although Rh110-labelled siRNA phosphorothioate (PTO)-modified ON (TM6-6) entered into ECV304 cells [96], similar results could not be obtained with either the human T-lymphoma cell line Jurkat 17 [97] or the mouse B-lymphoid cell line BJA-B [98]. In the absence of PTOs, the uptake of the siRNA was reduced and the molecules distributed throughout the cytoplasm [99].…”

“…However, when entry of siRNA has been demonstrated, there is an apparent dose-dependent siRNA-mediated suppression of lamin A/C in primary human umbilical vein endothelial cells [96,99]. Perhaps more importantly, Skog et al [81] showed that mRNA and miRNA can be taken up by normal host cells including brain and microvascular endothelial cells.…”

Phosphorothioate Nucleotides and Oligonucleotides – Recent Progress in Synthesis and Application

“…This discovery and strongly related phenomenon of synthetic short interfering RNA duplexes (siRNA) are thought to be promising tools for gene silencing. Interestingly, phosphorothioate oligonucleotides (in which one of the nonbridging phosphate O-atoms is replaced with a S-atom [20] (PS-Oligos; 2), were found to stimulate cellular uptake of naked siRNAs into mammalian cells, and this indicated a promising mechanistic strategy for a new delivery systems [21]. Recent progress in this dynamically developed area has been compiled in many reviews [22 -25].…”

“…A first method for stereochemically controlled chemical synthesis of PS-Oligos, which was elaborated by Stec et al [91], is based on a quite different chemistry employing P-diastereoisomerically pure nucleoside monomers possessing at the 3'-O-position the 2-thioxo-1,3,2-oxathiaphospholane moiety (19; Scheme 3), alternatively substituted at C(4) with either two Me groups (20) [92] or pentamethylene ring in a spiro arrangement (21) [93]. These substituents enhance a differentiation in chromatographic mobility of diastereoisomers, rendering their separation less laborious.…”