COPII vesicles can affect the activity of antisense oligonucleotides by facilitating the release of oligonucleotides...

Area of Science:

• Cell Biology
• Molecular Biology
• Drug Delivery

Background:

• Phosphorothioate antisense oligonucleotides (PS-ASOs) are used to modulate gene expression.
• PS-ASOs enter cells via endocytosis and must escape endosomes for therapeutic activity.
• Release from late endosomes (LEs) is a critical, yet poorly understood, step for PS-ASO efficacy.

Purpose of the Study:

• To elucidate the mechanism by which PS-ASOs are released from late endosomes.
• To identify cellular components involved in PS-ASO endosomal escape.
• To understand the role of COPII vesicles and associated proteins in PS-ASO release.

Main Methods:

• Cellular assays to monitor PS-ASO uptake and localization.
• Knockdown of COPII coat proteins and associated factors (STX5, P115).
• Confocal microscopy to assess co-localization of PS-ASOs with endosomal markers and COPII vesicles.

Main Results:

• COPII vesicles, typically involved in ER-Golgi transport, re-localized to PS-ASO-containing LEs.
• Reduction of COPII proteins decreased PS-ASO activity and slowed endosomal release, without affecting uptake.
• STX5 protein re-localized to LEs, bound PS-ASOs, and was crucial for COPII recruitment and PS-ASO release.

Conclusions:

• PS-ASO incubation triggers a novel pathway involving STX5-mediated recruitment of COPII vesicles to LEs.
• This pathway facilitates the release of PS-ASOs from endosomes, enhancing their antisense activity.
• STX5 is identified as a key protein binding PS-ASOs and mediating their escape from LEs.