A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery

Area of Science:

• Cell Biology
• Biotechnology
• Molecular Biology

Background:

• Extracellular vesicles (EVs) are key mediators of intercellular communication via cargo transfer.
• Understanding EV cargo loading and delivery is crucial for therapeutic applications.
• Current knowledge of general rules governing EV cargo loading and delivery is limited.

Purpose of the Study:

• To investigate the impact of biophysical properties on RNA loading and delivery by EVs.
• To develop a platform for actively loading engineered RNA into EVs.
• To identify limitations in EV-mediated mRNA and protein delivery to prostate cancer cells.

Main Methods:

• Engineered cargo RNAs with MS2 stem loops and fused MS2 coat protein to EV-associated proteins.
• Developed the Targeted and Modular EV Loading (TAMEL) platform for active RNA loading.
• Utilized vesicular stomatitis virus glycoprotein (VSVG)-expressing vesicles (gesicles) for enhanced loading studies.
• Assessed cargo RNA loading efficiency for different RNA sizes.
• Investigated EV-mediated delivery of mRNA and protein to prostate cancer cells, analyzing cargo degradation and uptake.

Main Results:

• The TAMEL platform achieved up to 6-fold enhanced cargo RNA loading into EVs.
• Gesicles showed a 40-fold enrichment in cargo RNA loading compared to standard EVs.
• Active loading was more efficient for smaller (~0.5 kb) RNA molecules than mRNA-length (>1.5 kb) ones.
• Despite high EV loading and uptake, most cargo was rapidly degraded in recipient prostate cancer cells.
• Inefficient endosomal fusion or escape was identified as a likely barrier to EV-mediated transfer in this model.

Conclusions:

• The TAMEL platform is a versatile tool for enhancing EV cargo loading and studying EV-mediated transfer.
• Significant improvements in EV cargo loading were achieved, particularly with gesicles.
• Cargo degradation in recipient cells limits the efficacy of EV-mediated delivery.
• Targeting endosomal escape or fusion is a key opportunity for enhancing EV-based therapeutics.