Overview
The video outlines a process for creating genetically modified CAR T cells through the CRISPR-Cas9 System. Infecting T cells with CRISPR and CAR lentiviruses results in modifications to the target gene and the synthesis of a chimeric antigen receptor or CAR, ultimately leading to the formation of genetically modified CAR T cells.
Protocol
1. CART19 cell production
- T-cell isolation, stimulation, and ex-vivo culture
- Carry out all cell culture work in a cell culture hood utilizing appropriate personal protective equipment. Harvest peripheral blood mononuclear cells (PBMCs) from de-identified normal donor blood cones collected during apheresis as these are known to be a viable source of PBMCs.
- To isolate PBMCs, add 15 mL of a density gradient medium to a 50 mL density gradient separation tube. Dilute the donor blood with an equal volume of phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) to avoid cell trapping.
- Add the diluted donor blood from the cone into the separation tube, careful not to disturb the interface between the blood and the density gradient medium. Centrifuge at 1,200 x g for 10 min at RT.
- Decant the supernatant into a new 50 mL conical, wash with PBS + 2% FBS by bringing up to 40 mL, centrifuge at 300 x g for 8 min at RT, aspirate supernatant, resuspend in 40 mL of buffer, and count cells.
- Isolate T cells from PBMCs via a negative selection magnetic bead kit using a fully automated cell separator according to the manufacturer's protocol.
- To culture the isolated T-cells, prepare T-cell medium (TCM) that consists of 10% human AB serum (v/v), 1% penicillin-streptomycin-glutamine (v/v), and serum-free hematopoietic cell medium. Sterilize the medium by filtering through a 0.45 µm sterile vacuum filter and then with a 0.22 µm sterile vacuum filter.
- On Day 0 (the day of T-cell stimulation), wash CD3/CD28 beads prior to stimulation of T-cells. To wash, place the required volume of beads (use enough CD3/CD28 beads for a ratio of 3:1 beads:T cells; note the concentration of beads can be variable) in a sterile 1.5 mL microcentrifuge tube and resuspend in 1 mL of TCM.
- Place in contact with a magnet.
- After 1 min, aspirate the TCM and resuspend in 1 mL of fresh TCM to wash the beads.
- Repeat this procedure for a total of three washes.
- After the third wash, resuspend the beads in 1 mL of TCM.
- Count the T-cells.
- Transfer beads to the T cells at a ratio of 3:1 beads:cells.
- Dilute cells to a final concentration of 1 x 106 cells/mL.
- Incubate at 37 °C, 5% CO2 for 24 h.
- T cell transfection and transduction
- Carry out lentiviral work using BSL-2+ precautions, including cell culture hoods, personal protective equipment, and disinfection of used materials with bleach before disposal.
- Acquire a chimeric antigen receptor-T19 (CART19) construct in a lentiviral vector.
NOTE: The CART19 construct utilized here was designed and then synthesized de novo using a commercially available protein synthesis vendor. The CAR construct was subsequently cloned into a third-generation lentivirus under control of an EF-1α promoter. The single chain variable region fragment is derived from the FMC63 clone and recognizes human CD19. The CAR19 construct possesses a second generation 4-1BB costimulatory domain and CD3ζ stimulation (FMC63-41BBz). - Perform lentiviral production as previously described.
- In brief, to produce lentivirus, utilize 293T-cells that have reached 70-90% confluency.
- Allow incubation for 30 min at room temperature of transfection reagents including 15 µg of the lentiviral plasmid of interest, 18 µg of a gag/pol/tat/rev packaging vector, 7 µg of a VSV-G envelope vector, 111 µL of the pre-complexing reagent, 129 µL of the transfection reagent, and 9.0 mL of the transfection medium before adding to the 293T-cells. Then culture the transfected cells at 37 °C, 5% CO2.
- Harvest, centrifuge (900 x g for 10 min), filter (0.45 µM nylon filter), and concentrate supernatant at 24 and 48 h by ultracentrifugation at either 13,028 x g for 18 h or 112,700 x g for 2 h.
- Freeze at -80 °C for future use.
- On Day 1, gently resuspend T-cells to break up the rosettes of T-cells that had been stimulated at 1 x 106/mL on Day 0.
- Under appropriate BSL-2+ precautions for all lentiviral work, add fresh or frozen harvested virus to the stimulated T-cells at a multiplicity of infection (MOI) of 3.0.
- CAR T-cell expansion
- During the phase of expansion, continue to incubate the transduced T-cells at 37 °C, 5% CO2. Count CAR T-cells on days 3 and 5, and add fresh, pre-warmed TCM to the culture to maintain a CAR T-cell concentration of 1 x 106/mL.
- Remove beads from the transduced T-cells 6 days after stimulation (Day 6) using a magnet. Harvest, resuspend, and place T cells in 50 mL conical tubes in a magnet for 1 min. Then collect the supernatant (contains the CAR T-cells), and discard the beads.
- Place the collected CAR T-cells back in culture at a concentration of 1 x 106 cells/mL to resume expansion.
- On Day 6, assess surface expression of the CAR by flow cytometry.
NOTE: Several methods can be used to detect the surface expression of CAR, such as staining with a goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody or by staining with a CD19 specific peptide, conjugated to a fluorochrome. Here, take an aliquot (about 100,000 T-cells) from the culture and wash with flow buffer prepared with Dulbecco's phosphate-buffered saline, 2% fetal bovine serum, and 1% sodium azide. Stain the cells with the anti-CAR antibody and wash twice. Stain the cells with live/dead stain and CD3 monoclonal antibody (OKT3). Wash the cells and resuspend in flow buffer. Acquire on a cytometer to determine transduction efficiency.
- CAR T-cell cryopreservation
- To harvest and cryopreserve CAR T-cells 8 days after stimulation (Day 8 of T cell expansion), harvest the cells from culture.
- Spin down for 5 min at 300 x g.
- Resuspend in freezing medium at 10 million cells per mL per vial in freezing medium consisting of 10% dimethyl sulfoxide and 90% fetal bovine serum.
- Freeze in a freezing container to achieve a rate of cooling of -1 °C/min in a -80 °C freezer and then transfer to liquid nitrogen after 48 h.
- Prior to their use for in vitro or in vivo experiments, thaw CAR T-cells in warm TCM.
- Wash the cells to dilute and remove the dimethyl sulfoxide and resuspend to a concentration of 2 x 106 cells/mL in warm TCM. Rest overnight at 37 °C, 5% CO2.
2. Granulocyte macrophage colony-stimulating factor (GM-CSF k/o) CART19 production
- To disrupt GM-CSF, utilize a guide RNA (gRNA) targeting exon 3 of human GM-CSF (CSF2) selected via screening gRNAs previously reported to have high efficiency for the CSF2 gene that encodes for the human cytokine GM-CSF.
NOTE: A commercially synthesized research grade Cas9 third generation lentiviral construct containing this gRNA (under a U6 promoter) was used. The construct contains a puromycin resistance gene. The sequence of the gRNA is GACCTGCCTACAGACCCGCC. - To produce lentivirus, utilize 293T cells that have reached 70-90% confluency.
- Allow 15 µg of the lentiviral plasmid of interest, 18 µg of a gag/pol/tat/rev packaging vector, 7 µg of a VSV-G envelope vector, 111 µL of the pre-complexing reagent, 129 µL of the transfection reagent, and 9.0 mL of the transfection medium to incubate for 30 min at room temperature.
- Add transfection reagents to the 293T-cells. The culture at 37 °C, 5% carbon dioxide (CO2).
- Harvest, centrifuge (900 x g for 10 min), filter (0.45 µM nylon filter), and concentrate supernatant at 24 and 48 h by ultracentrifugation at either 13,028 x g for 18 h or 112,700 x g for 2 h and freeze at -80 °C for future use.
- On Day 1, gently resuspend the T cells to break up rosettes.
- In a BSL-2+ approved laboratory, add a frozen or freshly harvested virus to the stimulated T-cells to generate CAR T-cells. Transduce T-cells with both the CAR19 lentivirus and GM-CSF targeting CRISPR lentivirus. Add CAR19 lentivirus at a MOI of 3. Since titration of the CRISPR lentivirus was not feasible, use virus particles generated from a 15 ug plasmid preparation to transduce 10 x 106 T cells.
- See the remaining steps of T-cell stimulation, expansion, and cryopreservation as discussed in step 1.
- For lentiCRISPR-edited T-cells carrying puromycin resistance, treat cells with puromycin dihydrochloride at a concentration of 1 µg of puromycin per 1 mL on Day 3 and Day 5.
Subscription Required. Please recommend JoVE to your librarian.
Materials
Name | Company | Catalog Number | Comments |
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 | GenScript | N/A | Custom order |
CRISPR guide RNA 1; Species: Human, Vector: | |||
pLentiCRISPR v2; Resistance: Ampicillin; Copy number: | |||
High; Plasmid preparation: Standard delivery: 4 μg (Free | |||
of charge) | |||
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 | 7851 | |
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. | 210055210 | |
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 |