Efficient in vivo genome editing mediated by stem cells-derived extracellular vesicles carrying designer nucleases

Abstract

Precise genome editing using designer nucleases (DNs), such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system, has become a method of choice in a variety of biological and biomedical applications in recent years. Notably, efficacy of these systems is currently under scrutiny in about 50 clinical trials. Although high DNs activity in various cell types in vitro has already been achieved, efficient in vivo genome editing remains a challenge. To solve this problem, we employed stem cells-derived extracellular vesicles (EVs) as carriers of DNs. We used umbilical cord-derived mesenchymal stem cells (UC-MSCs) and induced pluripotent stem cells (iPSCs) as EV-producer cells, since they are both applied in regenerative medicine. In our proof-of-concept studies, we achieved up to 50% of EGFP marker gene knockout in vivo using EVs carrying either ZFN, TALEN or the CRISPR/Cas9 system, particularly in the liver. Importantly, we obtained almost 50% of modified alleles in the liver of the experimental animals, when targeting the Pcsk9 gene, whose overexpression is implicated in hypercholesterolemia. Taken together, our data provide strong evidence that stem cells-derived EVs constitute a robust tool in delivering DNs in vivo, which may be harnessed to clinical practice in the future.

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“…The EV delivery method is gaining interest in science because of their natural potential to carry macromolecules such as Cas9 components. Bobis-Wozowicz et al showed the proof of concept that programable nucleases can be packaged into EVs for genomic target editing ( 136 ). Gee et al also demonstrated that EVs can be used as a delivery method for the CRISPR/Cas9 protein and sgRNA for exon 45 skipping in DMD induced pluripotent stem cells with 90% efficiency ( 137 ).…”

“…EVs represent a potential alternative for gene therapy cargo delivery. EVs are naturally produced by the human body and can be used to transfer large molecules ( 136 ).…”