Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells

Summary

The article describes a protocol for the application of an in vitro model for exhaustion to complete a genome-wide CRISPR knockout screen in healthy donor chimeric antigen receptor T cells.

Abstract

Chimeric antigen receptor T (CART) cell therapy is an innovative form of targeted immunotherapy that has revolutionized the treatment of cancer. However, the durable response remains limited. Recent studies have shown that the epigenetic landscape of preinfusion CART cell products can influence response to therapy, and gene editing has been proposed as a solution. However, more work needs to be done to determine the optimal gene editing strategy. Genome-wide CRISPR screens have become popular tools to both investigate mechanisms of resistance and optimize gene editing strategies. Yet their application to primary cells presents many challenges. Here we describe a method to complete a genome-wide CRISPR knockout screen in CART cells from healthy donors. As a proof-of-concept model, we designed this method to investigate the development of exhaustion in CART cells targeting the CD19 antigen. However, we believe that this approach can be used to address a variety of mechanisms of resistance to therapy in different CAR constructs and tumor models.

Introduction

Chimeric antigen receptor T (CART) cell therapy has shown impressive success in the treatment of B-cell malignancies; however, the durable response is limited to 30-40%^1,2,3,4,5. While researchers have developed and tested several approaches to address mechanisms of resistance to CART cell therapy, including the optimization of CAR design, gene editing, and combination therapies, the development of resistance remains largely unknown. Recently, there has been increasing evidence that the baseline gene expres....

Protocol

Importantly, the protocol outlined below follows guidelines from and has received approval from the Mayo Clinic's Institutional Review Board (IRB 18-005745) and the Institutional Biosafety Committee (IBC HIP00000252.43). All cell culture work, including lentiviral production, should be carried out in a cell culture hood with appropriate personal protective equipment. In particular, lentiviral work should be conducted under biosafety level 2 (BSL-2) precautions, including the use of 10% bleach to disinfect items befor.......

Discussion

Gene editing has become a powerful tool in both understanding the mechanisms of resistance to therapies as well as designing novel CART cell therapies to improve the longevity and activity of CART cells^16,17,26. While some gene editing strategies have shown improvements in CART cell activity in both preclinical models and clinical trials, there is still work to be done to optimize gene editing strategies. To address this need, r.......

Materials

Name	Company	Catalog Number	Comments
293T cells	ATCC	CRL-3216	Cells used for lentivirus production
Biotin ProteinL Antibody	GenScript	M00097	anti-kappa chain antibody for CAR detection
Bovine Serum Albumin	Millipore Sigma	A7906
Carbenicillin disodium salt	Millipore Sigma	C1389-1G	Carbenicillin antibiotic
CD4 Isolation Beads	Miltenyi Biotec	130-045-101
CD8 Isolation Beads	Miltenyi Biotec	130-045-201
CTS (Cell Therapy Systems) Dynabeads CD3/CD28	Gibco	40203D
Cytoflex	Beckman Coulter	NC2279958
DNase-Free Water	Invitrogen	AM9937
Dulbecco's modified eagle's medium (DMEM)	Corning	10-017-CV
Dulbecco's Phosphate-Buffered Saline	Gibco	14190-144
EasySep Human T Cell Isolation Kit	STEMCELL Technologies	17951RF	Negative isolation kit
Endura Electrocompetent Cells	Biosearch Technologies	60242-1	Electrocompetent cells with recovery medium
Ethanol	Millipore Sigma	E7023
Fetal bovine serum (FBS)	Corning	35-010-CV
GeCKO v2 CRISPR Knockout Pooled Library A	AddGene	1000000048	CRISPR library plasmid
Gene Pulser II	Bio-Rad	165-2105	Electroporator
Glycogen	Millipore Sigma	10901393001
JeKo-1	ATCC	CRL-3006	CD19+ target cells
Lipofectamine 3000 Transfection Reagent	ThermoFisher Scientific	L3000075	Transfection reagent kit with a transfection reagent (Lipofectamine 3000 Reagent) and a neutral co-lipid reagent (p3000)
LIVE/DEAD Aqua	Invitrogen	L34966
Lymphoprep	STEMCELL Technologies	7851	Density gradient medium
Machery-Nagel NucleoBond Xtra Maxi Kits	ThermoFisher Scientific	12748412	Maxi-prep kit
NEBNext High-Fidelity 2X PCR MasterMix	New England BioLabs	M0541S	High fidelity PCR mastermix
Opti-MEM I Reduced Serum Medium	Gibco	31985-070	Reduced serum medium
pCMVR8.74	AddGene	22036	Lentiviral packaging plasmid
Pennicillin-streptomycin-glutamine (100X)	Life Technologies	10378-016
pMD2.G	AddGene	12259	VSV-G envelope expressing plasmid
Pooled Human AB Serum	Innovative Research	ISERABHI
Puromycin	Millipore Sigma	P8833
QIAquick Gel Extraction Kit	Qiagen	28704	Gek extraction kit
Qucik-DNA Midiprep Plus Kit	Zymo Research	D4075	Kit used to isolate gDNA
RoboSep-S	STEMCELL Technologies	21000	Automated cell separator
Roswell Park Memorial Institute 1640 Medium (RPMI)	Gibco	21870092
SepMate-50	STEMCELL Technologies	85450	Density gradient separation tube
Sodium Acetate	Invitrogen	AM9740
Sodium Azide	Fisher Scientific	71448-16
Streptavidin Antibody (PE)	BioLegend	405203	Secondary antibody used for CAR detection
T100 Thermal Cycler	Bio-Rad	1861096
Ultracentrifuge (Optima XPN-80)	BeckmanCoulter	A99839
Vacuum Filter Systems, 0.22 µm	ThermoFisher Scientific	567-0020
Vacuum Filter Systems, 0.45 µm	ThermoFisher Scientific	165-0045
X-VIVO 15 Serum-Free Hematopoietic Cell Medium	Lonza	04-418Q	Hematopoietic cell medium