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Immunology and Infection

Production of Human CRISPR-Engineered CAR-T Cells

Published: March 15th, 2021



1Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, 2Parker Institute for Cancer Immunotherapy, University of Pennsylvania
* These authors contributed equally

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....

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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.......

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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.......

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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.......

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We acknowledge the Human Immunology Core for providing normal donor T cells and the Flow Cytometry Core at University of Pennsylvania.


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Name Company Catalog Number Comments
4D-Nucleofactor Core Unit Lonza AAF-1002B
4D-Nucleofactor X-Unit Lonza AAF-1002X
Accuprime Pfx Supermix ThermoFisher 12344040
Beckman Optima XPN ultracentrifuge Beckman Coulter
Brilliant Violet 605 anti-human CD3 Antibody Biolegend 317322 Clone OKT3
BV711 Anti-human PD1 Biolegend Clone EH12.2H7
Cas9-Electroporation enhancers IDT 1075915
CD3/CD28 Dynabeads ThermoFisher 40203D
CD4+ T cell isolation Kit StemCell technologies 15062
CD8+ T cell isolation Kit StemCell technologies 15063
Corning 0.45 micron vacuum filter/bottle Corning 430768
Corning T150 cell culture flask Millipore Sigma CLS430825
DMSO Millipore Sigma D2650
DNAeasy Blood and Tissue Kit Qiagen 69504
DynaMag Magnet ThermoFisher 12321D
Glutamax supplement ThermoFisher 35050061
HEK293T cells ATCC CRL-3216
HEPES (1 M) ThermoFisher 15630080
huIL-15 PeproTech 200-15
huIL-7 PeproTech 200-07
Lipofectamine 2000 ThermoFisher 11668019
Nucleospin Gel and PCR cleanup Takara 740609.25
Opti-MEM ThermoFisher 31985062
P3 Primary cell 4D-nucleofactor X Kit L Lonza V4XP-3024
Penicilin-Streptomycin-Glutamine ThermoFisher 10378016
pTRPE expression Plasmid in house
Rabbit Anti-Mouse FMC63 scFv Monoclonal Antibody, (R19M), PE CytoArt 200105
RPMI1640 ThermoFisher 12633012
Spy Fi Cas9 Aldevron 9214
Ultracentrifuge tubes Beckman Coulter 326823
Viral packaging mix in house
X-Vivo-15 Media Lonza BE02-060F

  1. Kowolik, C. M., et al. CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Research. 66 (22), 10995-11004 (2006).
  2. Krause, A., et al. Antigen-dependent CD28 signaling selectively enhances survival and proliferation in genetically modified activated human primary T lymphocytes. The Journal of Experimental Medicine. 188 (4), 619-626 (1998).
  3. Kuwana, Y., et al. Expression of chimeric receptor composed of immunoglobulin-derived V resions and T-cell receptor-derived C regions. Biochemical and biophysical research Communications. 149 (3), 960-968 (1987).
  4. Maher, J., Brentjens, R. J., Gunset, G., Rivière, I., Sadelain, M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRζ/CD28 receptor. Nature Biotechnology. 20 (1), 70-75 (2002).
  5. Levine, B. L., Miskin, J., Wonnacott, K., Keir, C. Global manufacturing of CAR-T cell therapy. Molecular Therapy-Methods & Clinical Development. 4, 92-101 (2017).
  6. Neelapu, S. S., et al. Axicabtagene ciloleucel CAR-T-cell therapy in refractory large B-cell lymphoma. New England Journal of Medicine. 377 (26), 2531-2544 (2017).
  7. Porter, D. L., et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Science Translational Medicine. 7 (303), 139 (2015).
  8. Maude, S. L., et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. New England Journal of Medicine. 371 (16), 1507-1517 (2014).
  9. Melero, I., Rouzaut, A., Motz, G. T., Coukos, G. T-cell and NK-cell infiltration into solid tumors: a key limiting factor for efficacious cancer immunotherapy. Cancer Discovery. 4 (5), 522-526 (2014).
  10. Mohammed, S., et al. Improving chimeric antigen receptor-modified T cell function by reversing the immunosuppressive tumor microenvironment of pancreatic cancer. Molecular Therapy. 25 (1), 249-258 (2017).
  11. Moon, E. K., et al. Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clinical Cancer Research. 20 (16), 4262-4273 (2014).
  12. Beatty, G. L., O'Hara, M. Chimeric antigen receptor-modified T cells for the treatment of solid tumors: defining the challenges and next steps. Pharmacology & Therapeutics. 166, 30-39 (2016).
  13. Stadtmauer, E. A., et al. CRISPR-engineered T cells in patients with refractory cancer. Science. 367 (6481), (2020).
  14. MacLeod, D. T., et al. Integration of a CD19 CAR into the TCR alpha chain locus streamlines production of allogeneic gene-edited CAR-T cells. Molecular Therapy. 25 (4), 949-961 (2017).
  15. Ren, J., et al. Multiplex genome editing to generate universal CAR-T cells resistant to PD1 inhibition. Clinical Cancer Research. 23 (9), 2255-2266 (2017).
  16. Ureña-Bailén, G., et al. CRISPR/Cas9 technology: towards a new generation of improved CAR-T cells for anticancer therapies. Briefings in Functional Genomics. 19 (3), 191-200 (2020).
  17. Li, C., Mei, H., Hu, Y. Applications and explorations of CRISPR/Cas9 in CAR-T-cell therapy. Briefings in Functional Genomics. 19 (3), 175-182 (2020).
  18. Ran, F. A., et al. Genome engineering using the CRISPR-Cas9 system. Nature Protocols. 8 (11), 2281-2308 (2013).
  19. Cong, L., et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 339 (6121), 819-823 (2013).
  20. Jinek, M., et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337 (6096), 816-821 (2012).
  21. Jinek, M., et al. RNA-programmed genome editing in human cells. Elife. 2, 00471 (2013).
  22. Schluns, K. S., Kieper, W. C., Jameson, S. C., Lefrançois, L. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nature Immunology. 1 (5), 426-432 (2000).
  23. Prlic, M., Lefrancois, L., Jameson, S. C. Multiple choices: regulation of memory CD8 T cell generation and homeostasis by interleukin (IL)-7 and IL-15. The Journal of Experimental Medicine. 195 (12), 49-52 (2002).
  24. Zhou, J., et al. Chimeric antigen receptor T (CAR-T) cells expanded with IL-7/IL-15 mediate superior antitumor effects. Protein & Cell. 10 (10), 764-769 (2019).
  25. Hultquist, J. F., et al. CRISPR-Cas9 genome engineering of primary CD4+ T cells for the interrogation of HIV-host factor interactions. Nature Protocols. 14 (1), 1-27 (2019).
  26. Brinkman, E. K., Chen, T., Amendola, M., van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Research. 42 (22), 168 (2014).

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