Cas9 ribonucleoprotein electroporation

100 pmol Alt-R crRNA and 100 pmol Alt-R tracrRNA (IDT) were diluted in IDT Duplex Buffer, incubated at 90° C for 5 min, and then slow cooled to room temperature (Tables S1, S7, and S8). 50 pmol S. pyogenes Cas9 V3 (IDT) was diluted in RNP buffer (20 mM HEPES, 150 mM NaCl, 10% Glycerol, 1 mM MgCl2, pH 7.5). Cas9 and duplexed gRNA (1:2 molar ratio) were incubated at 37° C for 15 min. Primary human T cells were washed once with PBS (-/-) before 250,000 cells were resuspended in P3 Buffer (Lonza). 50 pmol Cas9 RNP was added to the cells before electroporation in a Lonza 4D-Nucleofector with pulse code EH-115. X-VIVO 15 medium with 300 U/mL IL-2 was added to the electroporated cells before a 30 min recovery at 37° C. Electroporated T cells were plated at a density of 0.5-1x10^6 cells/mL in 96-well U-bottom plates.

CAR T cell production

gRNAs targeting exon 1 of the TRAC locus (TRAC gRNA 12), the intron preceding the TRAC locus (TRAC gRNA 13), or a non-targeting control gRNA were purchased from Synthego and resuspended in TE buffer (Table S11). Cas9 RNP was generated by incubating 60 pmol of Cas9 protein with 120 pmol sgRNA. T cells were counted, resuspended in P3 buffer at 2x10^6 per 20 mL, mixed with 3 mL of RNPs and added to a 96-well nucleofection plate. Cells were electroporated using a Lonza 4D-Nucleofector 96-well unit with the EH-115 protocol and immediately recovered by adding pre-warmed X-VIVO 15 medium without human serum.

Activated T cell versus non-activated T cell manufacturing

For the activated T cell editing protocol, T cells were activated and stimulated identical to what was described earlier. After electroporation of Cas9 RNP, T cells were cultured in X-VIVO 15 medium with 5 ng/mL IL-7, 5 ng/mL IL-15, and 300 U/mL IL-2. For the non-activated T cell editing protocol, we followed a protocol similar to what was used in our phase 1 clinical trial. After T cell isolation, cells were cultured in X-VIVO 15 medium with 5 ng/mL IL-7 and 5 ng/mL IL-15 for two days. Non-activated T cells were electroporated with 50 pmol Cas9 RNP using a Lonza 4D-Nucleofector with pulse code EH-115. After electroporation, cells were incubated in X-VIVO 15 medium with 5 ng/mL IL-7 and 5 ng/mL IL-15 for a 30 min recovery at 37° C. Electroporated T cells were plated at a density of 0.5-1x10^6 cells/mL in 96-well U-bottom plates. Two days after electroporation, cells were counted and activated/stimulated with a 1:1 ratio of anti-human CD3/CD28 magnetic Dynabeads to cells, as well as 5 ng/mL IL-7, 5 ng/mL IL-15, and 300 U/mL IL-2 for an additional 3 days.