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CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice

AuthorsMeca-Cortés, Óscar ; Guerra-Rebollo, Marta; Garrido, Cristina; Borrós, Salvador; Rubio, Núria ; Blanco, Jerónimo
Keywordsbystander suicide therapy
cell therapy
CRISPR/Cas9 knockin
mesenchymal stem cells
non-invasive bioluminescence imaging
Issue Date15-Aug-2017
CitationMolecular Therapy - Nucleic Acids 8: 395-403 (2017)
AbstractThe use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs), using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker) and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene), under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells. © 2017 The Author(s)
Publisher version (URL)This work was supported by MINECO/FEDER (grants SAF2015-64927-C2-1-R and SAF2015-64927-C2-2-R ) and the Instituto de Salud Carlos III (Red Temática de Investigación Cooperativa en Terapia Celular-TERCEL) . We would like to thank M.A. Pastor and C. Bestard (flow cytometry) for their excellent technical assistance.
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