Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells

Kath J, Du W, Pruene A, Braun T, Thommandru B, Turk R, Sturgeon ML, Kurgan GL, Amini L, Stein M, Zittel T, Martini S, Ostendorf L, Wilhelm A, Akyüz L, Rehm A, Höpken UE, Pruß A, Künkele A, Jacobi AM, Volk HD, Schmueck-Henneresse M, Stripecke R, Reinke P, Wagner DL
Source: Mol Ther Methods Clin Dev.
Publication Date: (2022)
Issue: 25: 311-330
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
Culture Media:
4D-Nucleofector® 96-well Systems
4D-Nucleofector® X-Unit

For electroporation of 1 × 10^6 cells, 20 µL of resuspended cells were transferred to 1.88 µL of RNP/HDRT (except during DNA-escalation studies where different volumes were used) and mixed thoroughly. Afterward, the T cell/RNP/HDRT mixture was transferred onto a 16-well electroporation strip (20 µL = 106 cells per well, Lonza) or an electroporation cartridge (100 µL = 5 × 10^6 cells, Lonza). Cells were carefully transferred onto the electroporation strips using 200-µL tips to avoid trapping air in the solution. Electroporation strips and cartridges were tapped onto the bench several times to ensure correct placement of fluids within the electroporation vessel. Electroporation was performed on a 4D-Nucleofector Device (Lonza) using the program EH-115. Directly after electroporation, pre-warmed T cell medium was added onto the cells (90 µL per well and 450 µL per cartridge). Afterward, resuspended cells were transferred to 96-well round-bottom plates (50 µL/well) containing 150 µL pre-warmed T cell medium per well (with or without HDR-enhancing supplements) at a density of 0.5 × 10^6 cells per well.

For large-scale CAR T cell generation by electroporation in cartridges, 950 µL of pre-warmed T cell medium was used for initial resuspension. Ten minutes after electroporation, T cells were transferred into 24-well flat-bottom plates (500 µL/well) containing 1.5 mL of T cell medium (with or without HDR-enhancing supplements) at a density of 2.5 × 10^6 cells per well.

Jurkat cells (Clone E6-1, ATCC TIB-152) were cultured in RPMI media (ATCC) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% Pen Strep (Gibco), maintained at a density between 10^5 and 10^6 cells/mL. Cas9 gRNA targeting HPRT1 was prepared by mixing equimolar amounts of Alt-R CRISPR-RNA (crRNA) and Alt-R trans-activating CRISPR RNA (tracrRNA) (IDT, Coralville, IA, USA) in Tris-EDTA (TE), heating to 95°C, and slow cooling at room temperature. A single-stranded DNA oligo consisting of 40 nt of homology flanking the Cas9 cleavage site and a 6-base EcoRI (5'-GAATTC-3') restriction site inserting at the Cas9 cleavage site was designed (Table S3); the non-targeting (guide-containing) sequence strand was ordered as an Alt-R HDR Donor Oligo (IDT). The RNP was formed by complexation of 56-pmol IDT Cas9 protein with 67.2-pmol gRNA complex in a total volume of 7.5 µL with PBS to adjust to the final volume. All reagents were delivered to Jurkat cells using the Lonza Nucleofector System. Cells were counted and pelleted by centrifugation (300g, 10 min at room temperature) and washed with 10 mL 1× PBS. The cells were again pelleted and resuspended in Lonza Nucleofection Solution SE at a density of 1 × 10^8 cells/mL. For each electroporation, 5 µL of RNP was added to 20 µL of Jurkat cells in Nucleofection Solution SE (5 × 10^5 cells/nucleofection). Donor DNA and IDT Alt-R Cas9 Electroporation Enhancer were added to achieve a final concentration of 1 and 3 µM, respectively, in a total reaction volume of 28 µL. The solution was mixed by pipetting, and 25 µL was transferred to an electroporation cuvette plate. The cells were electroporated according to the manufacturer’s protocol using the Lonza 96-well Shuttle and nucleofection protocol 96-CL-120. 


Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant (TRAC) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome-editing method for efficient CAR insertion into the TRAC locus of primary human T cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knockin rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knockin rates and CAR T cell yield. Resulting TRAC-replaced CD19-CAR T cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair-modulating small molecules. With TRAC-integrated CAR+ T cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T cells.