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Genome editing of donor-derived aβ+ T cells
Nalm6-GL and Raji (GFP-Luciferase transduced) as target cells. After co-culture for 20 h, we were able to measure production of interleukin-2 and interferon-g in the cell culture supernatant specifically for cells with integration of the CAR (Figure 3A). To estimate the fraction of target cells that was killed, we determined the counts of target
A
cells (identified by their GFP expression) after 20 h of co- culture with either CAR T cells or control cells relative to samples cultured without effector cells, which showed cytotoxic activity even at low effector-to-target (E:T) ratios (Figure 3B).
To determine the fate of the B cells, which are part of
BCD
EFGH
Figure 2. Targeted integration of a CD19-specific chimeric antigen receptor into the TRAC locus. (A) Targeting strategy using Cas9 ribonucleoprotein and recombi- nant adeno-associated virus serotype 6. (B) Representative fluorescence activated cell sorting (FACS) plots for cells treated as indicated 4 days after targeting to evaluate the efficiency of T-cell receptor (TCR)aβ knockout and nerve growth factor receptor (NGFR) expression. (C) Quantification of the populations after targeting of T cells from 11 different donors. (D) Representative FACS plot of the cells stained for NGFR and a CD19-CAR idiotype-specific antibody. (E) FACS plot showing NGFR and TCRaβ expression after depletion of cells expressing the aβ T-cell receptor. (F) Quantification of aβ TCR depletion efficiency for four different replicates, plotted as mean ± standard deviation. (G) Expansion of T cells during the 7 days after gene editing compared to numbers before electroporation using optimized conditions for adeno-associated virus transduction. (H) Expansion of T cells (compared to numbers before gene editing) cultured at different densities after electroporation. RNP: ribonucleoprotein; rAAV6: recombinant adeno-associated virus serotype 6; NGFR: nerve growth factor receptor; pA: poly-adenylation signal; 2A: 2A peptide.
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