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CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy

T cells engineered with chimeric antigen receptors (CARs) show great promise in the treatment of some cancers. Modifying T cells to express CARs generally relies on T-cell transduction using viral vectors carrying a transgene, resulting in semi-random DNA integration within the T-cell genome. While...

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Autores principales: Odé, Zelda, Condori, Jose, Peterson, Nicolas, Zhou, Sheng, Krenciute, Giedre
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352666/
https://www.ncbi.nlm.nih.gov/pubmed/32604839
http://dx.doi.org/10.3390/cancers12061704
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author Odé, Zelda
Condori, Jose
Peterson, Nicolas
Zhou, Sheng
Krenciute, Giedre
author_facet Odé, Zelda
Condori, Jose
Peterson, Nicolas
Zhou, Sheng
Krenciute, Giedre
author_sort Odé, Zelda
collection PubMed
description T cells engineered with chimeric antigen receptors (CARs) show great promise in the treatment of some cancers. Modifying T cells to express CARs generally relies on T-cell transduction using viral vectors carrying a transgene, resulting in semi-random DNA integration within the T-cell genome. While this approach has proven successful and is used in generating the Food and Drug Administration (FDA, USA) approved B-lymphocyte antigen CD19-specific CAR T cells, it is possible the transgene could integrate into a locus that would lead to malignant transformation of the engineered T cells. In addition, manufacturing viral vectors is time-consuming and expensive. One way to overcome these challenges is site-specific gene integration, which can be achieved through clustered regularly interspaced short palindromic repeat (CRISPR) mediated editing and non-viral DNA, which serves as a template for homology-directed repair (HDR). This non-viral gene editing approach provides a rapid, highly specific, and inexpensive way to engineer T cells. Here, we describe an optimized protocol for the site-specific knock-in of a large transgene in primary human T cells using non-viral double stranded DNA as a repair template. As proof-of-principle, we targeted the T-cell receptor alpha constant (TRAC) locus for insertion of a large transgene containing green fluorescence protein (GFP) and interleukin-15 (IL-15). To optimize the knock-in conditions we tested template DNA concentration, homology arm length, cell number, and knock-in efficiency over time. We then applied these established guidelines to target the TRAC or interleukin-13 (IL-13) locus for the knock-in of synthetic molecules, such as a CAR, bispecific T-cell engager (BiTE), and other transgenes. While integration efficiency depends on the targeted gene locus and selected transgene, this optimized protocol reliably generates the desired insertion at rates upwards of 20%. Thus, it should serve as a good starting point for investigators who are interested in knocking in transgenes into specific loci.
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spelling pubmed-73526662020-07-21 CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy Odé, Zelda Condori, Jose Peterson, Nicolas Zhou, Sheng Krenciute, Giedre Cancers (Basel) Article T cells engineered with chimeric antigen receptors (CARs) show great promise in the treatment of some cancers. Modifying T cells to express CARs generally relies on T-cell transduction using viral vectors carrying a transgene, resulting in semi-random DNA integration within the T-cell genome. While this approach has proven successful and is used in generating the Food and Drug Administration (FDA, USA) approved B-lymphocyte antigen CD19-specific CAR T cells, it is possible the transgene could integrate into a locus that would lead to malignant transformation of the engineered T cells. In addition, manufacturing viral vectors is time-consuming and expensive. One way to overcome these challenges is site-specific gene integration, which can be achieved through clustered regularly interspaced short palindromic repeat (CRISPR) mediated editing and non-viral DNA, which serves as a template for homology-directed repair (HDR). This non-viral gene editing approach provides a rapid, highly specific, and inexpensive way to engineer T cells. Here, we describe an optimized protocol for the site-specific knock-in of a large transgene in primary human T cells using non-viral double stranded DNA as a repair template. As proof-of-principle, we targeted the T-cell receptor alpha constant (TRAC) locus for insertion of a large transgene containing green fluorescence protein (GFP) and interleukin-15 (IL-15). To optimize the knock-in conditions we tested template DNA concentration, homology arm length, cell number, and knock-in efficiency over time. We then applied these established guidelines to target the TRAC or interleukin-13 (IL-13) locus for the knock-in of synthetic molecules, such as a CAR, bispecific T-cell engager (BiTE), and other transgenes. While integration efficiency depends on the targeted gene locus and selected transgene, this optimized protocol reliably generates the desired insertion at rates upwards of 20%. Thus, it should serve as a good starting point for investigators who are interested in knocking in transgenes into specific loci. MDPI 2020-06-26 /pmc/articles/PMC7352666/ /pubmed/32604839 http://dx.doi.org/10.3390/cancers12061704 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Odé, Zelda
Condori, Jose
Peterson, Nicolas
Zhou, Sheng
Krenciute, Giedre
CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy
title CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy
title_full CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy
title_fullStr CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy
title_full_unstemmed CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy
title_short CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy
title_sort crispr-mediated non-viral site-specific gene integration and expression in t cells: protocol and application for t-cell therapy
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352666/
https://www.ncbi.nlm.nih.gov/pubmed/32604839
http://dx.doi.org/10.3390/cancers12061704
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