Immunology and Infection
Published: March 15th, 2021
Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.
Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.
Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the field of adoptive cell therapies and cancer immunotherapy. CAR-T-cells are engineered T-cells expressing a synthetic immune receptor that combines an antigen-specific single chain antibody fragment with signaling domains derived from the TCRzeta chain and costimulatory domains necessary and sufficient for T-cell activation and co-stimulation1,2,3,4. The manufacturing of CAR-T cells starts by extracting the patient's own T-cells, followed by ex vivo viral transduction o....
Human T cells were procured through the University of Pennsylvania Human Immunology Core, which operates under principles of Good Laboratory Practice with established standard operating procedures and/or protocols for sample receipt, processing, freezing, and analysis conform to MIATA and University of Pennsylvania ethics guidelines.
1. Lentiviral vector production
NOTE: The viral products have been made replication-defective by separation of packaging constructs (Rev.......
We describe here a protocol to genetically engineer T cells, that can be used to generate both autologous and allogeneic CAR-T cells, as well as TCR redirected T cells.
Figure 1 provides a detailed description of the stages involved in the process of manufacturing CRISPR edited T cells. The process begins by designing sgRNA to the gene of interest. Once the sgRNA are designed and synthesized they are then used to make RNP complexes with the appropriate Cas protein.......
Here we describe approaches to gene edit CAR-T cells using CRISPR Cas9 technology and manufacture products to further test for function and efficacy. The above protocol has been optimized for performing CRIPSR gene editing in primary human T cells combined with engineering T cells with chimeric antigen receptors. This protocol allows high knockout efficiency with minimal donor-to-donor variability. Modification using CRISPR can improve both the efficacy and safety of CAR-T cells by eliminating receptors that inhibit T ce.......
|4D-Nucleofactor Core Unit
|Accuprime Pfx Supermix
|Beckman Optima XPN ultracentrifuge
|Brilliant Violet 605 anti-human CD3 Antibody
|BV711 Anti-human PD1
|CD4+ T cell isolation Kit
|CD8+ T cell isolation Kit
|Corning 0.45 micron vacuum filter/bottle
|Corning T150 cell culture flask
|DNAeasy Blood and Tissue Kit
|HEPES (1 M)
|Nucleospin Gel and PCR cleanup
|P3 Primary cell 4D-nucleofactor X Kit L
|pTRPE expression Plasmid
|Rabbit Anti-Mouse FMC63 scFv Monoclonal Antibody, (R19M), PE
|Spy Fi Cas9
|Viral packaging mix
Copyright © 2024 MyJoVE Corporation. All rights reserved