Targeted insertion of a CD19-specific CAR into the IL-2 gene-by-gene editing produces highly functional T cells in vitro and in vivo. (A-B) IL-2–sgRNA:Cas9-mediated cleavage of the human interleukin-2 (hIL-2) protein. The edited and control T cells after 3-day electroporation were treated with protein kinase C activator TPA and calcium ionophore A23187 for 45 minutes to induce IL-2 production, followed by the overnight addition of brefeldin A or dimethyl sulfoxide (control) to block IL-2 secretion before collecting supernatant and lysing the cells. Intracellular and supernatant IL-2 protein levels were detected by western blot (A) and ELISA (B). LV-CAR and KI-CAR T cells represent transduced and edited CAR T cells, respectively. Data are presented as mean ± standard deviation (SD) (n = 3; a paired t test). (C) A diagram of CRISPR/Cas9-targeted CAR19 gene integration into the IL-2 locus. Upper portion: sgRNA-targeting sequence (green), PAM sequence (red), ATG (underline), and a vertical arrowhead for cleavage site. Middle portion: the genomic organization of the human IL-2 gene with exons represented by boxes and coding domains (cyan). Lower portion: donor DNA sequence containing an LHA (white), a promoter (gray), 19BBζ CAR coding sequence, polyA, and RHA (cyan) sequence (supplemental Table 6). (D) Representative flow cytometry showing the kinetics of nonintegrated and integrated CAR expression from nanoplasmid-encoded CAR transgene. TransAct-stimulated T cells were transfected with nanoplasmids with or without the sgRNA-Cas9 complex, followed by flow cytometry for checking CAR expressions at days 3, 6, 10, and 13 postelectroporation. Flow cytometry analysis showed that nonintegrated CAR expression was eliminated on day 6, whereas integrated CAR expression remained detectable on day 10 postelectroporation. (E) CAR MFI and CAR CV of CAR+ T cells (n = 12 independent experiments). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 (Welch's 2 samples t test). (F) The killing potential of the IL-2–targeted CAR (KI-CAR) T cells vs transduced CAR T cells (LV-CAR T) was evaluated using xCELLigence RTCA eSight. Cytotoxicity against green fluorescent protein (GFP)-expressing Nalm6-CD19 target cells was evaluated at effector-to-target ratios (E:T) of 1:1 and 0.5:1 as depicted in the figure. Unedited T cells (mock) and target cells with or without 0.1% Triton X-100 (100% lysis control) were used as controls. The percentage of target cell death was quantified based on the confluency of GFP-labeled target cells followed by CAR T-cell addition. (G) The percentage of target cell death was quantified using the AUC of GFP-labeled target cells following CAR T-cell addition. E:T ratio = 1:1. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001 Statistical significance was determined by repeated measures 1-way analysis of variance (ANOVA) with multiple comparisons (n = 3). (H) ELISA detection of IFN-γ and TNF-α in the cultured supernatants of CAR T cocultured with Nalm6-CD19 cells for 24 hours at an E:T ratio of 2:1. Data represent the mean ± SD of 3 independent donors. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Repeated measures 1-way ANOVA (multiple comparisons) (n = 3). (I) The evaluation of the antitumor efficacy of KI-CAR19 T cells in vivo using a mouse model of leukemia (Nalm6). The individual BLI intensity of Nalm6-LUC was determined for each mouse of KI-CAR19 and transduced CAR19 (LV-CAR T) groups (n = 5 mice per group). (J) The quantification of the total human CD45+, CAR+ T-cell, CD4+, and CD8+ T-cell subsets in the mice of the transduced or edited groups were assayed on day 14 after CAR T-cell infusion. The data are presented as mean ± SD (n = 5 mice per group). Groups were compared using paired t tests. (K) The quantification of TN (naïve), TCM (central memory), TEM (effector memory), and TEMRA (effector memory RA) cell subpopulations of the transduced or edited groups on day 14 after CAR T-cell infusion. The data are presented as mean ± SD (n = 5 mice per group). Groups were compared using the Mann-Whitney test. ATG, adenine-thymine-guanine; AUC, area under the curve; CV, coefficient of variance; hIL-2, human interleukin-2; IFN-γ, interferon gamma; KI-CAR T, knockin chimeric antigen receptor T-cell; LHA, left homology arm; MFI, mean fluorescence intensity; RHA, right homology arm; TNF-α, tumor necrosis factor α.

Targeted insertion of a CD19-specific CAR into the IL-2 gene-by-gene editing produces highly functional T cells in vitro and in vivo. (A-B) IL-2–sgRNA:Cas9-mediated cleavage of the human interleukin-2 (hIL-2) protein. The edited and control T cells after 3-day electroporation were treated with protein kinase C activator TPA and calcium ionophore A23187 for 45 minutes to induce IL-2 production, followed by the overnight addition of brefeldin A or dimethyl sulfoxide (control) to block IL-2 secretion before collecting supernatant and lysing the cells. Intracellular and supernatant IL-2 protein levels were detected by western blot (A) and ELISA (B). LV-CAR and KI-CAR T cells represent transduced and edited CAR T cells, respectively. Data are presented as mean ± standard deviation (SD) (n = 3; a paired t test). (C) A diagram of CRISPR/Cas9-targeted CAR19 gene integration into the IL-2 locus. Upper portion: sgRNA-targeting sequence (green), PAM sequence (red), ATG (underline), and a vertical arrowhead for cleavage site. Middle portion: the genomic organization of the human IL-2 gene with exons represented by boxes and coding domains (cyan). Lower portion: donor DNA sequence containing an LHA (white), a promoter (gray), 19BBζ CAR coding sequence, polyA, and RHA (cyan) sequence (supplemental Table 6). (D) Representative flow cytometry showing the kinetics of nonintegrated and integrated CAR expression from nanoplasmid-encoded CAR transgene. TransAct-stimulated T cells were transfected with nanoplasmids with or without the sgRNA-Cas9 complex, followed by flow cytometry for checking CAR expressions at days 3, 6, 10, and 13 postelectroporation. Flow cytometry analysis showed that nonintegrated CAR expression was eliminated on day 6, whereas integrated CAR expression remained detectable on day 10 postelectroporation. (E) CAR MFI and CAR CV of CAR+ T cells (n = 12 independent experiments). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 (Welch's 2 samples t test). (F) The killing potential of the IL-2–targeted CAR (KI-CAR) T cells vs transduced CAR T cells (LV-CAR T) was evaluated using xCELLigence RTCA eSight. Cytotoxicity against green fluorescent protein (GFP)-expressing Nalm6-CD19 target cells was evaluated at effector-to-target ratios (E:T) of 1:1 and 0.5:1 as depicted in the figure. Unedited T cells (mock) and target cells with or without 0.1% Triton X-100 (100% lysis control) were used as controls. The percentage of target cell death was quantified based on the confluency of GFP-labeled target cells followed by CAR T-cell addition. (G) The percentage of target cell death was quantified using the AUC of GFP-labeled target cells following CAR T-cell addition. E:T ratio = 1:1. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001 Statistical significance was determined by repeated measures 1-way analysis of variance (ANOVA) with multiple comparisons (n = 3). (H) ELISA detection of IFN-γ and TNF-α in the cultured supernatants of CAR T cocultured with Nalm6-CD19 cells for 24 hours at an E:T ratio of 2:1. Data represent the mean ± SD of 3 independent donors. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Repeated measures 1-way ANOVA (multiple comparisons) (n = 3). (I) The evaluation of the antitumor efficacy of KI-CAR19 T cells in vivo using a mouse model of leukemia (Nalm6). The individual BLI intensity of Nalm6-LUC was determined for each mouse of KI-CAR19 and transduced CAR19 (LV-CAR T) groups (n = 5 mice per group). (J) The quantification of the total human CD45+, CAR+ T-cell, CD4+, and CD8+ T-cell subsets in the mice of the transduced or edited groups were assayed on day 14 after CAR T-cell infusion. The data are presented as mean ± SD (n = 5 mice per group). Groups were compared using paired t tests. (K) The quantification of TN (naïve), TCM (central memory), TEM (effector memory), and TEMRA (effector memory RA) cell subpopulations of the transduced or edited groups on day 14 after CAR T-cell infusion. The data are presented as mean ± SD (n = 5 mice per group). Groups were compared using the Mann-Whitney test. ATG, adenine-thymine-guanine; AUC, area under the curve; CV, coefficient of variance; hIL-2, human interleukin-2; IFN-γ, interferon gamma; KI-CAR T, knockin chimeric antigen receptor T-cell; LHA, left homology arm; MFI, mean fluorescence intensity; RHA, right homology arm; TNF-α, tumor necrosis factor α.

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