Genetically Modifying CAR T Cells Using a CRISPR-Cas9 System

Protocol

1. CART19 cell production

1. T-cell isolation, stimulation, and ex-vivo culture
   1. 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.
   2. 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.
      1. 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.
      2. 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.
   3. Isolate T cells from PBMCs via a negative selection magnetic bead kit using a fully automated cell separator according to the manufacturer's protocol.
   4. 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.
   5. 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.
   6. Place in contact with a magnet.
   7. After 1 min, aspirate the TCM and resuspend in 1 mL of fresh TCM to wash the beads.
   8. Repeat this procedure for a total of three washes.
   9. After the third wash, resuspend the beads in 1 mL of TCM.
   10. Count the T-cells.
   11. Transfer beads to the T cells at a ratio of 3:1 beads:cells.
   12. Dilute cells to a final concentration of 1 x 10⁶ cells/mL.
   13. Incubate at 37 °C, 5% CO₂ for 24 h.
2. T cell transfection and transduction
   1. Carry out lentiviral work using BSL-2+ precautions, including cell culture hoods, personal protective equipment, and disinfection of used materials with bleach before disposal.
   2. 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).
   3. Perform lentiviral production as previously described.
      1. In brief, to produce lentivirus, utilize 293T-cells that have reached 70-90% confluency.
      2. 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.
      3. 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.
      4. Freeze at -80 °C for future use.
   4. 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.
   5. 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.
3. CAR T-cell expansion
   1. 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.
   2. 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.
   3. Place the collected CAR T-cells back in culture at a concentration of 1 x 10⁶ cells/mL to resume expansion.
   4. 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.
4. CAR T-cell cryopreservation
   1. To harvest and cryopreserve CAR T-cells 8 days after stimulation (Day 8 of T cell expansion), harvest the cells from culture.
   2. Spin down for 5 min at 300 x g.
   3. 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.
   4. 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.
   5. Prior to their use for in vitro or in vivo experiments, thaw CAR T-cells in warm TCM.
   6. 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

1. 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.
2. To produce lentivirus, utilize 293T cells that have reached 70-90% confluency.
3. 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.
4. Add transfection reagents to the 293T-cells. The culture at 37 °C, 5% carbon dioxide (CO₂).
5. 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.
6. On Day 1, gently resuspend the T cells to break up rosettes.
7. 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.
8. See the remaining steps of T-cell stimulation, expansion, and cryopreservation as discussed in step 1.
9. 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.

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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