Generation of CAR-T Cells with Sleeping Beauty Transposon Gene Transfer.
Adoptive cell therapy
Cancer
Chimeric antigen receptor
Gene transfer
Genetic vectors
Genomic integration
Immunotherapy
Transposon
Vector copy number
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
22
6
2022
pubmed:
23
6
2022
medline:
25
6
2022
Statut:
ppublish
Résumé
Human T lymphocytes that transgenically express a chimeric antigen receptor (CAR) have proven efficacy and safety in gene- and cell-based immunotherapy of certain hematological cancers. Appropriate gene vectors and methods of genetic engineering are required for therapeutic cell products to be biologically potent and their manufacturing to be economically viable. Transposon-based gene transfer satisfies these needs, and is currently being evaluated in clinical trials. In this protocol we describe the basic Sleeping Beauty (SB) transposon vector components required for stable gene integration in human cells, with special emphasis on minicircle DNA vectors and the use of synthetic mRNA. We provide a protocol for functional validation of the vector components in cultured human cell lines on the basis of fluorescent reporter gene expression. Finally, we provide a protocol for CAR-T cell engineering and describe assays that address transgene expression, biological potency and genomic vector copy numbers in polyclonal cell populations. Because transposons allow virus-free gene transfer with naked nucleic acids, the protocol can be adopted by any laboratory equipped with biological safety level S1 facilities.
Identifiants
pubmed: 35732992
doi: 10.1007/978-1-0716-2441-8_3
doi:
Substances chimiques
DNA Transposable Elements
0
Receptors, Antigen, T-Cell
0
Receptors, Chimeric Antigen
0
Transposases
EC 2.7.7.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
41-66Informations de copyright
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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