CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice
Author
Meca Cortés, Oscar
Guerra Rebollo, Marta
Garrido, Cristina
Borrós i Gómez, Salvador
Rubio, Nuria
Blanco, Jeronimo
Other authors
Universitat Ramon Llull. IQS
Publication date
2017-07-26ISSN
2162-2531
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.
Document Type
Article
Document version
Published version
Language
English
Subject (CDU)
573 - General and theoretical biology
615 - Pharmacology. Therapeutics. Toxicology
Keywords
CRISPR/Cas9
Cell therapy
Mesenchymal stem cells
Bystander suicide therapy
Glioblastoma
Non-invasive bioluminescence imaging
CRISPR/Cas9 knockin
CRISPR-Cas9
CRISPR (Genètica)
Teràpia cel·lular
Cèl·lules mare mesenquimàtiques
Glioblastoma multiforme
Imatgeria per al diagnòstic
CRISPR (Genètica)
Pages
9 p.
Publisher
Cell Press
Is part of
Molecular Therapy: Nucleic Acids
Grant agreement number
info:eu-repo/grantAgreement/MINECO i FEDER/PN I+D/SAF2015-64927-C2-1-R
info:eu-repo/grantAgreement/MINECO i FEDER/PN I+D/SAF2015-64927-C2-2-R
This item appears in the following Collection(s)
Rights
© L'autor/a
Except where otherwise noted, this item's license is described as http://creativecommons.org/licenses/by-nc-nd/4.0/