Small extracellular vesicles (sEVs)-based gene delivery platform for cell-specific CRISPR/Cas9 genome editing

Area of Science:

• Biotechnology
• Nanomedicine
• Molecular Biology

Background:

• Small extracellular vesicles (sEVs) show promise as drug delivery systems.
• Current sEV applications face challenges in cargo loading and targeting specificity.

Purpose of the Study:

• To develop a modular sEV-based platform (safeEXO) for efficient and targeted delivery of gene editing tools.
• To engineer safeEXO vesicles for in vitro and in vivo CRISPR-Cas9 delivery and genome editing.

Main Methods:

• Developed the modular safeEXO platform, engineered to be RNA-depleted.
• Incorporated Cas9 and guide RNA into safeEXO vesicles for CRISPR-RNP delivery.
• Engineered safeEXO vesicles with integrin alpha-6 for lung-specific targeting.
• Administered targeted safeEXO-CAS-ITGA6 vesicles with EMX1 sgRNAs to mice for lung gene editing.

Main Results:

• safeEXO vehicles efficiently delivered RNA and RNP complexes intracellularly.
• safeEXO-CAS vesicles mediated dose-dependent CRISPR genome editing in vitro and in vivo.
• Targeted safeEXO-CAS-ITGA6 vesicles enhanced lung epithelial cell uptake and induced lung-specific gene editing in mice.
• No significant toxicity or immune response was observed in treated mice.

Conclusions:

• The modular safeEXO platform is a safe, targetable, and effective vehicle for nucleic acid therapeutics delivery.
• Genetically engineered producer cells enable production of sEVs loaded with CRISPR machinery for enhanced genome editing.
• This platform holds potential for advancing RNAi and CRISPR-based therapies for human diseases.