Prime editing of IL-2Rβ can be combined with IL-2 knockin for vector-free generation of oIL-2–responsive CAR T cells. (A) A schematic representation of PE-ortho/KI-CAR19 editing strategy. The first step is generating KI-CAR T cells. The IL-2–targeting sgRNA and Cas9 protein were incubated for 10 to 15 minutes at room temperature before adding donor template, followed by the addition of the TransAct-activated human primary T cells in P3 buffer. The mixture was transferred into the Lonza cuvettes and strip wells followed by electroporation using the pulse code EH115, resulting in the generation of KI-CAR T cells. After 3 to 4 days, PE2 mRNA, pegRNA, and ngRNA were cotransfected into KI-CAR T cells using the same 4D-Nucleofector system (Lonza Bioscience) using the pulse code E0115, enabling the expression of oIL-2Rβ. (B) NSG mice were engrafted with 1 × 10⁶ Nalm6 cells on day 0 and received 1 × 10⁶ PE-ortho/KI-CAR19 T cells or nonedited cells (mock) as a control on day 5 following BLI on day 4. The mice with PE-ortho/KI-CAR19 T cells were divided into 2 groups: phosphate-buffered saline (red line) and oIL-2 (dark blue line). A total of 20 000 IU of oIL-2 was administered through intraperitoneal injection once a day for 21 days. Tumor burden was assessed by BLI twice per week (n = 5 mice per group). (C) The quantification of bioluminescence values of the phosphate-buffered saline-treated and oIL-2–treated groups. Comparison of BLI intensity between phosphate-buffered saline and oIL-2 treatment groups was performed using the Mann-Whitney test. ∗P < .05; ∗∗P < .01. (D) The total quantification of PE-mediated oIL-2Rβ mutation in the mice receiving oIL-2 or phosphate-buffered saline (n = 3) after PE-edited oIL-2–CAR T-cell infusion. ∗∗∗P < .001, ∗∗∗∗P < .0001 (E) The total human CD45⁺, CAR⁺ T-cell, CD4⁺, and CD8⁺ T-cell subsets in the mice receiving oIL-2 or phosphate-buffered saline on day 14. The data are presented as mean ± SD (n = 5 mice per group). Groups were compared using the Mann-Whitney test. Red indicates the phosphate-buffered saline-treated PE-ortho/KI-CAR group; blue indicates the oIL-2–treated PE-ortho/KI-CAR group. (F) Mouse body weight of 3 different groups was normalized to the body weight on day 0 for each group over time until day 23. Light blue indicates the mock group; red indicates the phosphate-buffered saline-treated PE-ortho/KI-CAR group; blue indicates the orthoIL-2–treated PE-ortho/KI-CAR group. (G) The analysis of IFN-γ serum levels by ELISA in the phosphate-buffered saline-treated and oIL-2–treated mice on day 7 and 14 after T-cell infusion. The bars represent mean ± SD values with the range (n = 8). IFN-γ, interferon gamma; KI-CAR T, knockin chimeric antigen receptor T cell; ns, not significant; NSG, NOD scid gamma.