Production of Human CRISPR-Engineered CAR-T Cells

Summary

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

Abstract

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.

Introduction

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-stimulation^1,2,3,4. The manufacturing of CAR-T cells starts by extracting the patient's own T-cells, followed by ex vivo viral transduction o....

Protocol

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, gag/pol/RRE, VSVg and transfer plasmid) into four separate plasmids, greatly reducing the likelihood of recombination events that may result in repl....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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
sgRNA	IDT
Spy Fi Cas9	Aldevron	9214
Ultracentrifuge tubes	Beckman Coulter	326823
Viral packaging mix	in house
X-Vivo-15 Media	Lonza	BE02-060F