Optimized Cholesterol-siRNA Chemistry Improves Productive Loading onto Extracellular Vesicles

Area of Science:

• Biotechnology
• Molecular Biology
• Nanomedicine

Background:

• Extracellular vesicles (EVs) are natural carriers for therapeutic RNAs.
• Cholesterol conjugation facilitates RNA loading into EVs.
• Optimizing chemical modifications of siRNAs is crucial for efficient EV-mediated delivery.

Purpose of the Study:

• To evaluate the impact of chemical modifications on hydrophobic siRNAs (hsiRNAs) for EV loading.
• To compare different linkers for cholesterol conjugation to siRNAs.
• To determine optimal loading conditions for hsiRNAs in EVs.

Main Methods:

• Synthesized cholesterol-conjugated siRNAs with various backbone, 5'-phosphate, and linker modifications.
• Loaded hsiRNAs into EVs and assessed loading efficiency.
• Evaluated gene silencing efficacy of loaded EVs in neuronal cells (Huntingtin silencing).
• Determined siRNA loading capacity and saturation point of EVs.

Main Results:

• hsiRNAs with 5'-(E)-vinylphosphonate and alternating 2'-fluoro/2'-O-methyl backbone modifications showed superior EV loading and silencing.
• Triethyl glycol (TEG) linker was more effective than C7 for exosomal loading.
• Linker destabilization abolished silencing activity.
• EV loading saturated at approximately 3,000 siRNA copies per EV, with overloading impairing activity.

Conclusions:

• Hydrophobic modification is a viable strategy for productive RNA loading into EVs.
• Specific chemical modifications and linker selection are critical for optimizing EV-mediated RNA delivery.
• Understanding loading capacity prevents overloading and maintains therapeutic efficacy.