COPII vesicles can affect the activity of antisense oligonucleotides by facilitating the release of oligonucleotides from endocytic pathways

Abstract

RNase H1-dependent, phosphorothioate-modified antisense oligonucleotides (PS-ASOs) can enter cells through endocytic pathways and need to be released from the membrane-enclosed organelles, a limiting step for antisense activity. Accumulating evidence has suggested that productive PS-ASO release mainly occurs from late endosomes (LEs). However, how PS-ASOs escape from LEs is not well understood. Here, we report that upon PS-ASO incubation, COPII vesicles, normally involved in ER–Golgi transport, can re-locate to PS-ASO-containing LEs. Reduction of COPII coat proteins significantly decreased PS-ASO activity, without affecting the levels of PS-ASO uptake and early-to-late endosome transport, but caused slower PS-ASO release from LEs. COPII co-localization with PS-ASOs at LEs does not require de novo assembly of COPII at ER. Interestingly, reduction of STX5 and P115, proteins involved in tethering and fusion of COPII vesicles with Golgi membranes, impaired COPII re-localization to LEs and decreased PS-ASO activity. STX5 can re-locate to LEs upon PS-ASO incubation, can bind PS-ASOs, and the binding appears to be required for this pathway. Our study reveals a novel release pathway in which PS-ASO incubation causes LE re-localization of STX5, which mediates the recruitment of COPII vesicles to LEs to facilitate endosomal PS-ASO release, and identifies another key PS-ASO binding protein.

Differential uptake, kinetics and mechanisms of intracellular trafficking of next-generation antisense oligonucleotides across human cancer cell lines

“…To do this, we focused on NSCLC cells that are enriched for KRAS mutations occurring in ∼33% of lung adenocarcinoma (26). Cellular internalization of ASO was determined by immunofluorescence using an antibody that specifically detects the phosphorothioate backbone of the antisense drug (14,27). ASO accumulated within all the cells in a dose-dependent manner (Figure 2A and Supplementary Figure S5A), and even in cell lines with moderate or poor KRAS knockdown we observed ASO staining within the cell (Figure 2B and C).…”

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

“…It is more likely that the sequence context of the PS tail may be affecting efficiency of cellular interactions similar in mechanism to those reported for ASOs. 15 There are multiple articles describing functional pathways involved in productive internalization of ASOs inside the cells; while many protein co-factors are described (for example, lysobisphosphatidic acid, 56 Sec31A, 57 and Rab5 58 ), it is clear that productive interactions are redundant and knockout of any of the aforementioned pathways individually does not fully block productive silencing. Thus, complete functional characterization of molecular mechanisms behind PS-enhanced uptake is technically complex and will require further investigation.…”

Retinal proteomics of experimental glaucoma model reveal intraocular pressure‐induced mediators of neurodegenerative changes

“…46 We identified nine proteins associated with the intracellular SNARE transport complex to be differentially regulated in the retinas of high IOP exposed animals. Sec23b and Stx5 proteins which are involved in endoplasmic reticulum-Golgi transport 47 were observed to be upregulated (Figure 3). On the other hand, Importin proteins (IPO5 and IPO9) that mediate energy-dependent transportation through the nuclear pore, were downregulated 48 along with other SNARE proteins such as Cops8, Cplx2, Gorasp2, Manf, and Stx4.…”