Protein Kinase C-α is a Critical Protein for Antisense Oligonucleotide-mediated Silencing in Mammalian Cells

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Antisense oligonucleotides (ASOs) are therapeutic agents that silence gene expression.
• Understanding the intracellular trafficking and mechanisms of ASO action is crucial for optimizing their delivery and efficacy.
• Gymnotic delivery, a non-viral method, is an emerging strategy for introducing ASOs into cells.

Purpose of the Study:

• To identify and characterize the endocytotic pathway involved in gymnotically delivered ASO cellular uptake.
• To elucidate the role of protein kinase C-alpha (PKC-α) in ASO trafficking and gene silencing.
• To differentiate between cytoplasmic and nuclear gene silencing pathways mediated by ASOs.

Main Methods:

• Utilized locked nucleic acid (LNA)-gapmer phosphorothioate antisense oligonucleotides (ASOs) for gymnotic delivery.
• Investigated the role of PKC-α by silencing its expression and observing effects on ASO activity.
• Performed endosome maturation blocking experiments to track ASO localization.
• Conducted reconstitution experiments to restore PKC-α expression and assess ASO silencing ability.

Main Results:

• Identified a PKC-α-dependent endocytotic pathway that directs ASOs to late endosomes.
• Silencing PKC-α significantly reduced ASO-mediated gene silencing efficacy.
• Impaired endosome-to-late endosome maturation led to reduced ASO silencing.
• Cytoplasmic gene silencing by ASOs was found to be dependent on PKC-α expression.
• Restoration of PKC-α expression reinstated ASO silencing capabilities.

Conclusions:

• PKC-α plays a critical role in the intracellular trafficking and activity of gymnotically delivered ASOs.
• A distinct PKC-α-dependent endocytotic pathway is involved in ASO uptake and late endosome localization.
• These findings reveal the existence of at least two distinct gene silencing pathways in mammalian cells: cytoplasmic and nuclear.
• This study advances the understanding of ASO cellular mechanisms, informing the development of more effective oligonucleotide-based therapeutics.