TY - JOUR T1 - Efficient in vivo genome editing mediated by stem cells-derived extracellular vesicles carrying designer nucleases JF - bioRxiv DO - 10.1101/2021.02.25.432823 SP - 2021.02.25.432823 AU - Sylwia Bobis-Wozowicz AU - Karolina Kania AU - Kinga Nit AU - Natalia Blazowska AU - Katarzyna Kmiotek-Wasylewska AU - Milena Paw AU - Elzbieta Karnas AU - Agnieszka Szyposzynska AU - Malgorzata Tyszka-Czochara AU - Olga Woznicka AU - Dariusz Boruczkowski AU - Claudio Mussolino AU - Paweł P. Łabaj AU - Axel Schambach AU - Zbigniew Madeja AU - Toni Cathomen AU - Ewa K. Zuba-Surma Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/02/26/2021.02.25.432823.abstract N2 - 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.Competing Interest StatementJagiellonian University and Medical Center - University of Freiburg have filed a patent application for the use of stem cell-derived EVs as carriers of gene modifying enzymes on behalf of the inventors SB-W, EZ-S. and TC. TC has a funded research collaboration with Cellectis. The remaining authors declare no competing interests. This work was supported by the Homing Plus/2013-7/3 project carried out within the Homing Plus programme of the Foundation for Polish Science (FNP) co-financed by the European Union under the European Regional Development Fund, the SONATA12 project: UMO-2016/23/D/NZ3/01310 from the National Science Centre of Poland granted to S.B.-W., the project TEAM-2012/9-6 (FNP), POIG.01.01.02-00-109/09, STRATEGMED 3 (STRATEGMED3/303570/7/NCBR/2017) funded by National Center for Research and Development (NCBR) to E.Z.-S., and the support from the KNOW grant of the FBBB from the Ministry of Science and Higher Education of Poland. ER -