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

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101526829 Publication Model: Electronic Cited Medium: Print ISSN: 2072-6694 (Print) Linking ISSN: 20726694 NLM ISO Abbreviation: Cancers (Basel) Subsets: PubMed not MEDLINE

Original Publication: Basel, Switzerland : MDPI

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.

Cell. 2016 Oct 6;167(2):419-432.e16. (PMID: 27693353)
Cell. 2016 Feb 11;164(4):780-91. (PMID: 26830878)
Nat Rev Immunol. 2018 Oct;18(10):605-616. (PMID: 30046149)
Proc Natl Acad Sci U S A. 2015 Aug 18;112(33):10437-42. (PMID: 26216948)
Mol Ther. 2017 Apr 5;25(4):949-961. (PMID: 28237835)
Sci Rep. 2018 Aug 3;8(1):11649. (PMID: 30076383)
Nature. 2017 May 24;545(7655):423-431. (PMID: 28541315)
Cancer Cell. 2012 Feb 14;21(2):212-26. (PMID: 22340594)
Mol Ther. 2006 Jan;13(1):15-25. (PMID: 16260184)
Sci Rep. 2018 Jan 17;8(1):888. (PMID: 29343825)
Gene Ther. 2008 Jun;15(11):840-8. (PMID: 18418418)
Cytometry A. 2011 Aug;79(8):646-52. (PMID: 21695774)
Nat Commun. 2019 Nov 19;10(1):5222. (PMID: 31745080)
Mol Ther. 2016 Feb;24(2):354-363. (PMID: 26514825)
Sci Transl Med. 2014 Feb 19;6(224):224ra25. (PMID: 24553386)
Hum Mol Genet. 2016 Apr 15;25(R1):R42-52. (PMID: 26519140)
Blood. 2017 Jun 22;129(25):3322-3331. (PMID: 28408462)
N Engl J Med. 2014 Oct 16;371(16):1507-17. (PMID: 25317870)
Lancet. 2015 Feb 7;385(9967):517-528. (PMID: 25319501)
Genome Res. 2017 Jul;27(7):1099-1111. (PMID: 28356322)
Mol Ther. 2015 Jan;23(1):171-8. (PMID: 25142939)
Nat Biotechnol. 2020 Jan;38(1):44-49. (PMID: 31819258)
Mol Ther Methods Clin Dev. 2017 Jan 10;4:192-203. (PMID: 28345004)
Front Genet. 2019 Jan 07;9:691. (PMID: 30687381)
Nature. 2017 Mar 2;543(7643):113-117. (PMID: 28225754)
Proc Natl Acad Sci U S A. 2017 Dec 12;114(50):E10745-E10754. (PMID: 29183983)
Curr Gene Ther. 2016;16(3):156-67. (PMID: 27216914)
Nat Rev Cancer. 2011 Dec 01;12(1):51-8. (PMID: 22129804)
Hum Gene Ther. 2011 Mar;22(3):343-56. (PMID: 21043787)
EMBO Rep. 2017 Oct;18(10):1707-1715. (PMID: 28801534)
Cytotherapy. 2019 Dec;21(12):1198-1205. (PMID: 31837735)
Sci Rep. 2019 Mar 12;9(1):4194. (PMID: 30862905)
Nat Med. 2017 Apr 3;23(4):415-423. (PMID: 28388605)
Toxicol Sci. 2017 Feb;155(2):315-325. (PMID: 27803388)
Nature. 2018 Jul;559(7714):405-409. (PMID: 29995861)
Sci Rep. 2016 Feb 16;6:20889. (PMID: 26879144)
Science. 2003 Oct 17;302(5644):415-9. (PMID: 14564000)

n/a Assisi Foundation of Memphis

Keywords: CAR; CRISPR-Cas9; HDR; T cell; TRAC; knock-in; non-viral

Date Created: 20200702 Latest Revision: 20200928

20231215

PMC7352666

10.3390/cancers12061704

32604839