Our protocol describes the genetic modification of CAR-T cell during their manufacturing via the CRISPR technology to generate more effective and less toxic product. An advantage of this technique is that CAR-T cell production and genetic manipulation can both occur in one cell with good efficiency. Demonstrating the procedure will be Rosalie Sterner, an MD PhD student, Michelle Cox, a technologist and a graduate student, and Reona Sakemura, a postdoctorate fellow, from my laboratory.
After isolating T-cells from harvested peripheral blood mononuclear cells, start culturing them by first preparing T-cell medium and then sterilizing it by filtering it through a 0.45 micrometer sterile vacuum filter and then with a 0.22 micrometer sterile vacuum filter. On the day of T-cell stimulation or day zero, prior to stimulation, wash CD3/CD28 beads by placing the required volume of beads in a sterile 1.5 milliliter microcentrifuge tube and resuspending in one milliliter of TCM. Place the tube in contact with a magnet for one minute.
Then aspirate the TCM and resuspend in one milliliter of fresh TCM to wash the beads again and repeat for a total of three washes. And finally, resuspend the beads in one milliliter of TCM. After counting the T-cells, transfer the beads to the T-cells at a ratio of three to one beads to cells.
Then dilute the cells to a final concentration of one million cells per milliliter and incubate at 37 degrees Celsius, 5%CO2 for 24 hours. After acquiring a CART19 construct in a lentiviral vector, perform lentiviral production by first incubating lentiviral plasmid, packaging vector, envelope vector, precomplexing reagent, transfection reagent, and transfection medium at room temperature for 30 minutes. Carry out lentiviral work using BSL2+precautions including cell culture hoods, personal protective equipment, and disinfection of used materials with bleach before disposal.
After incubation, add these transfection reagents to the 293 T-cells that have reached 70 to 90%confluency and culture the transfected cells at 37 degrees Celsius and 5%CO2. At 24 and 48 hours after transfection, harvest, centrifuge, filter and concentrate virus-containing supernatant by ultracentrifugation and freeze at minus 80 degrees Celsius for future use. On day one, gently resuspend T-cells that have been stimulated at one million cells per milliliter on day zero in order to break up the rosettes of cells.
Under appropriate BSL2+precautions, add fresh or frozen harvested virus to the simulated T-cells at a multiplicity of infection of 3.0. Continue to incubate the transduced T-cells at 37 degrees Celsius, 5%CO2. On days three and five, count CAR-T cells and add fresh pre-warmed TCM to the culture to maintain a CAR-T cell concentration of one million cells per milliliter.
Six days after simulation, remove beads from the transduced T-cells by first harvesting and resuspending the cells. Transfer the cells in 50 milliliter conical tubes and place the tubes in a magnet for one minute. After collecting the CAR-T cell-containing supernatant and discarding the beads, place the cells back in culture at a concentration of one million cells per milliliter in a culture flask.
Use a sample of cells for flow cytometry to assess surface expression of the CARs. Incubate the rest to resume expansion and then harvest and cryo-preserve the cells at day eight as described in the manuscript. To disrupt the granulocyte macrophage colony stimulating factor, utilize a guide RNA as described in the manuscript.
Incubate transfection reagents at room temperature for 30 minutes. After incubation, add them to the 293 T-cells that have reached 70 to 90%confluency and culture the transfected cells at 37 degrees Celsius, 5%CO2 to produce a lentivirus. At 24 and 48 hours, harvest and concentrate virus-containing supernatant by ultracentrifugation in 50 milliliter ultracentrifuge tubes and freeze at minus 80 degrees Celsius for future use.
Then on day one, gently resuspend the T-cells to break up rosettes. In a BSL2+approved laboratory, to generate CAR-T cells, add CAR19 lentivirus and GMCSF-targeting CRISPR lentivirus to the simulated T-cells. On day three and day five for successfully transduced lentiCRISPR-edited T-cells carrying puromycin resistance, treat cells with puromycin dihydrochloride at a concentration of one microgram of puromycin per milliliter.
Tide sequencing was used to confirm a GM-CSF reduction in GM-CSF knockout CART19 cells with a disruption efficiency of approximately 71%Flow plots of CAR-T cell surface staining gated on live CD3-positive cells show that both wild-type CART19 and GM-CSF knockout CART19 successfully and in a similar fashion express the CAR surface receptor in vitro. On the other hand, intracellular staining of GM-CSF by flow cytometry also gated on live CD3-positive cells demonstrates decreased expression of GM-CSF in the knockout cells compared to wild-type CART19 cells confirming functional success of the knockout. Particular care should be taken during the transduction steps of this procedure as they are the most critical to the development of genetically modified CAR-T cells.
The methodology described can potentially be applied to a variety of genes to modify CAR-T cells via CRISPR/CAS9 to help generate a less toxic and more effective product. CRISPR/CAS9 technology provides the strategies to directly target the genome of CAR-T cells to engineer solutions to current clinical shortcomings.
