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Mol Ther Nucleic Acids. 2017 Sep 15;8:395-403. doi: 10.1016/j.omtn.2017.07.012. Epub 2017 Jul 18.

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

Author information

1
Cell Therapy Group, Catalonian Institute for Advanced Chemistry (IQAC-CSIC), Jordi Girona, 18-26, Barcelona 08034, Spain; Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Zaragoza 50018, Spain.
2
Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Zaragoza 50018, Spain; Grup d' Enginyeria de Materials (GEMAT), Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta, 390, Barcelona 08017, Spain.
3
Cell Therapy Group, Catalonian Institute for Advanced Chemistry (IQAC-CSIC), Jordi Girona, 18-26, Barcelona 08034, Spain; Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Zaragoza 50018, Spain. Electronic address: jbfnqb@iqac.csic.es.

Abstract

The 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.

KEYWORDS:

CRISPR/Cas9; CRISPR/Cas9 knockin; bystander suicide therapy; cell therapy; glioblastoma; mesenchymal stem cells; non-invasive bioluminescence imaging

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