Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.
Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.
Chimeric antigen receptor T (CAR-T) cell therapy exhibits great promise in the treatment of cancer.1,2 Two CAR-T cell therapies targeting CD19 (CART19) were recently approved in the United Stated and in Europe for the use in B cell malignancies after demonstrating striking results in multicenter clinical trials.3,4,5 Barriers to more widespread use of CAR-T cells are limited activity in solid tumors and associated toxicities including cytokine release syndrome (CRS) and neurotoxicity (NT).3,5,6,7,8,9 To enhance the therapeutic index of CAR-T cell therapy, genome engineering tools such as zinc finger nucleases, TALENs, and CRISPR are employed to further modify CAR-T cells in an attempt to generate less toxic or more effective CAR-T cells.10,11
In this article, we describe a method to generate CRISPR/Cas9 edited CAR-T cells. The specific goal of this method is to genetically modify CAR-T cells during CAR-T cell manufacturing via CRISPR/Cas9 to generate less toxic or more effective CAR-T cells. The rationale for developing this methodology is built on lessons learned from clinical experience of CAR-T cell therapy, which indicates an urgent need for novel strategies to increase the therapeutic window of CAR-T cell therapy and to extend the application into other tumors and is supported by the recent advances in synthetic biology allowing multiple modifications of CAR-T cells that have started to enter the clinic. While several genome engineering tools are being developed and applied in different settings, such as zinc finger nucleases, TALENs, and CRISPR, our methodology describes CRISPR/Cas9 modification of CAR-T cells.10,11 CRISPR/Cas9 is an RNA-based bacterial defense mechanism that is designed to eliminate foreign DNA. CRISPR relies on endonucleases to cleave a target sequence identified through a guide RNA (gRNA). CRISPR editing of CAR-T cells offers several advantages over other genome engineering tools. These include precision of the gRNA sequence, simplicity to design a gRNA targeting the gene of interest, high gene editing efficiency, and the ability to target multiple genes since multiple gRNAs can be used at the same time.
Specifically in the methods described here, we used a lentivirus encoding CRISPR guide RNA and Cas9 to disrupt a gene during CAR transduction of T cells. In selecting an appropriate technique to edit CAR-T cells, we suggest the technique described here is an efficient mechanism to generate research grade CAR-T cells, but because the long term effect of permanent integration of Cas9 into the genome is unknown, we propose this methodology to develop proof of concept research grade CAR-T cells but not for producing good manufacturing practice grade CAR-T cells.
In particular, here we describe the generation of granulocyte macrophage colony stimulating factor (GM-CSF) knockout CAR-T cells targeting human CD19. These CAR-T cells were generated by transduction with lentiviral particles encoding a guide RNA specific to GM-CSF (gene name CSF2) and Cas9. We previously found that GM-CSF neutralization ameliorates CRS and NT in a xenograft model.12 GM-CSFk/o CAR-T cells allow for the inhibition of GM-CSF during the manufacturing process, effectively reducing production of GM-CSF while enhancing CAR-T cell anti-tumor activity and survival in vivo compared to wildtype CAR-T cells.12 Thus, here we provide a methodology to generate CRISPR/Cas9 edited CAR-T cells.
In this report, we describe a methodology to utilize CRISPR/Cas9 technology to induce secondary modifications in CAR-T cells. Specifically, this is demonstrated using lentiviral transduction with a viral vector that contains gRNA targeting the gene of interest and Cas9 to generate GM-CSFk/o CART19 cells. We had previously shown that GM-CSF neutralization ameliorates CRS and NT in a xenograft model.12 As previously described, GM-CSFk/o CAR-T cells allow for the inhib…
The authors have nothing to disclose.
This work was supported through grants from K12CA090628 (SSK), the National Comprehensive Cancer Network (SSK), the Mayo Clinic Center for Individualized Medicine (SSK), the Predolin Foundation (SSK), the Mayo Clinic Office of Translation to Practice (SSK), and the Mayo Clinic Medical Scientist Training Program Robert L. Howell Physician-Scientist Scholarship (RMS).
CD3 Monoclonal Antibody (OKT3), PE, eBioscience | Invitrogen | 12-0037-42 | |
CD3 Monoclonal Antibody (UCHT1), APC, eBioscience | Invitrogen | 17-0038-42 | |
Choice Taq Blue Mastermix | Denville Scientific | C775Y51 | |
CTS (Cell Therapy Systems) Dynabeads CD3/CD28 | Gibco | 40203D | |
CytoFLEX System B4-R2-V2 | Beckman Coulter | C10343 | flow cytometer |
dimethyl sulfoxide | Millipore Sigma | D2650-100ML | |
Dulbecco's Phosphate-Buffered Saline | Gibco | 14190-144 | |
Dynabeads MPC-S (Magnetic Particle Concentrator) | Applied Biosystems | A13346 | |
Easy 50 EasySep Magnet | STEMCELL Technologies | 18002 | |
EasySep Human T Cell Isolation Kit | STEMCELL Technologies | 17951 | negative selection magnetic beads; 17951RF includes tips and buffer |
Fetal bovine serum | Millipore Sigma | F8067 | |
FITC Mouse Anti-Human CD107a | BD Pharmingen | 555800 | |
Fixation Medium (Medium A) | Invitrogen | GAS001S100 | |
GenCRISPR gRNA Construct: Name: CSF2 CRISPR guide RNA 1; Species: Human, Vector: pLentiCRISPR v2; Resistance: Ampicillin; Copy number: High; Plasmid preparation: Standard delivery: 4 μg (Free of charge) |
GenScript | N/A | custom order |
Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 | Invitrogen | A-21235 | |
https://tide.nki.nl. | Desktop Genetics | ||
Human AB Serum; Male Donors; type AB; US | Corning | 35-060-CI | |
IFN gamma Monoclonal Antibody (4S.B3), APC-eFluor 780, eBioscience | Invitrogen | 47-7319-42 | |
Lipofectamine 3000 Transfection Reagent | Invitrogen | L3000075 | |
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation | Invitrogen | L34966 | |
Lymphoprep | STEMCELL Technologies | 07851 | |
Monensin Solution, 1000X | BioLegend | 420701 | |
Mouse Anti-Human CD28 Clone CD28.2 | BD Pharmingen | 559770 | |
Mouse Anti-Human CD49d Clone 9F10 | BD Pharmingen | 561892 | |
Mouse Anti-Human MIP-1β PE-Cy7 | BD Pharmingen | 560687 | |
Mr. Frosty Freezing Container | Thermo Scientific | 5100-0001 | |
NALM6, clone G5 | ATCC | CRL-3273 | acute lymphoblastic leukemia cell line |
Nuclease Free Water | Promega | P119C | |
Olympus Vacuum Filter Systems, 500 mL, PES Membrane, 0.22uM, sterile | Genesee Scientific | 25-227 | |
Olympus Vacuum Filter Systems, 500 mL, PES Membrane, 0.45uM, sterile | Genesee Scientific | 25-228 | |
Opti-MEM I Reduced-Serum Medium (1X), Liquid | Gibco | 31985-070 | |
PE-CF594 Mouse Anti-Human IL-2 | BD Horizon | 562384 | |
Penicillin-Streptomycin-Glutamine (100X), Liquid | Gibco | 10378-016 | |
Permeabilization Medium (Medium B) | Invitrogen | GAS002S100 | |
PureLink Genomic DNA Mini Kit | Invitrogen | K182001 | |
Puromycin Dihydrochloride | MP Biomedicals, Inc. | 0210055210 | |
QIAquick Gel Extraction Kit | QIAGEN | 28704 | |
Rat Anti-Human GM-CSF BV421 | BD Horizon | 562930 | |
RoboSep-S | STEMCELL Technologies | 21000 | Fully Automated Cell Separator |
SepMate-50 (IVD) | STEMCELL Technologies | 85450 | |
Sodium Azide, 5% (w/v) | Ricca Chemical | 7144.8-16 | |
X-VIVO 15 Serum-free Hematopoietic Cell Medium | Lonza | 04-418Q |