Role of autophagy in cell-penetrating peptide transfection model

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Cell-penetrating peptides (CPPs) are crucial for delivering molecules into cells.
• The precise mechanism of CPP uptake and its cellular consequences remain incompletely understood.
• Oligonucleotide delivery via CPPs is a promising gene therapy approach requiring mechanistic insights.

Purpose of the Study:

• To investigate the early cellular events following PepFect14 (PF14) uptake in HeLa cells.
• To determine the impact of PF14, with or without oligonucleotide cargo, on gene expression.
• To elucidate the role of cellular pathways, particularly autophagy, in CPP-mediated transfection.

Main Methods:

• RNA sequencing and quantitative PCR (qPCR) to analyze gene expression profiles.
• Signaling pathway analysis to identify induced biological processes.
• Confocal and transmission electron microscopy to visualize cellular responses.
• Ligand library screening to probe the involvement of specific pathways.

Main Results:

• Early (1-hour) treatment revealed significant changes in RNA expression profiles.
• Autophagy-related genes were notably induced upon PF14-oligonucleotide complex uptake.
• Interference with autophagy pathways demonstrated concentration-dependent effects on transfection efficiency.
• Autophagy induction and colocalization with autophagosomes were confirmed via microscopy.

Conclusions:

• Cellular uptake of CPP-based transfection systems, like PF14, actively triggers the autophagy process.
• Autophagy is an inherent cellular response activated by the CPP delivery mechanism.
• Targeting autophagy modulation presents a novel strategy for optimizing future gene therapy applications.