Abstract

Engineered Extracellular Vesicles for CRISPR/Cas9 Gene Editing

Niek Dekker, Associate Director, Discovery Biology, AstraZeneca

Extracellular vesicles (EVs) represent an exciting opportunity as biological delivery vehicles for therapeutic cargo with excellent safety, low intrinsic immunogenicity, cell-specific tropism and biological delivery efficiency. There are multiple approaches for the introduction of protein and RNA cargo into EVs, including physical, chemical and cell engineering. Application of CRISPR/Cas9 requires the functional delivery of the ribonucleoprotein (RNP) complex of Cas9 enzyme and guide RNA. Engineering of the production cell line can be used to introduce cargo into the EVs, and examples for the introduction of fluorescent and luminescent proteins will be disclosed. Uptake in recipient cells can be monitored using high content imaging to provide insight in kinetics and localization. Functional delivery can be analyzed using several assays for the gene editing event (eg Cel1 and TIDE). The use of EVs for delivery of CRISPR/Cas9 has a range of exciting applications for ex vivo manipulation of primary cells and in vivo editing of genes in diseased tissue.