Abstract

Engineered Extracellular Vesicles For CRISPR Gene Editing

Niek Dekker, Associate Director, Discovery Biology, AstraZeneca

Exosomes present an exciting opportunity as biological delivery vehicle of therapeutic cargo with excellent safety, low intrinsic immunogenicity, cell-specific tropism and biological delivery efficacy. Application of CRISPR/Cas9 requires the functional delivery of the ribonucleoprotein (RNP) complex of Cas9 and guide RNA. Engineering of the production cell lines can be used to introduce cargo into the exosomes, and examples for the introduction of fluorescent and luminescent proteins in exosomes 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 (e.g., Cel1 and TIDE). Use of exosomes for delivery of CRISPR/Cas9 has applications for ex vivo manipulation of primary cells (e.g., T cells) and in vivo editing of genes in diseased tissue.