Fig. 1. Non-viral, AAVS1-integrated CAR-T cells effectively eliminate tumour cells.
a, Specific integration of the CAR cassette into the target locus by homologous recombination through CRISPR–Cas9. b,c, Percentage of CAR+ cells (b) and number of viable CAR+ cells (c) detected 7 d after electroporation using equimolar amounts of DNA templates with different homology arm lengths (n = 2 independent healthy donors). d, CAR expression in cells from two representative healthy donors determined 7 d after electroporation. SSC, side scatter. K, ×1,000. e, Percentage of CAR+ cells detected 7 d after electroporation (n = 23 independent healthy donors). f, Expansion of CAR+ cells after repeated stimulation with Raji cells. Data are shown as the mean ± s.e.m. (n = 3 independent healthy donors). g, Median fluorescence intensity (MFI) of CD69, CD137, CD25, PD1 and LAG3 expression in T cells detected after 24 h of co-culture with Raji cells (n = 3 independent healthy donors). CD3+ (untreated T, control) or CD3+CAR+ (LV-19bbz, AAVS1-19bbz) gated cells were analysed. h, Cytokine secretion measured by bead-based immunoassay in the supernatant after co-culture with Raji cells for 24 h. Data are shown as the mean ± s.e.m. (n = 3 independent healthy donors). IL-2, interleukin-2; TNFα, tumour necrosis factor α; IFNγ, interferon-γ. i, In vitro cytotoxicity against Raji cells as determined by lactate dehydrogenase (LDH) assay. E/T ratio, effector/target ratio. Data are shown as the mean ± s.e.m. (n = 3 independent healthy donors). j, Bioluminescence imaging of tumour cell growth following different treatments on the indicated days after CAR-T cell infusion (n = 5). The radiance scale (p s–1 cm–2 sr–1) is shown. Immunodeficient mice were injected intravenously with 2 × 105 firefly luciferase (ffLuc)-transduced Raji cells, and 2 × 106 CAR-T cells were administered intravenously after 5 d. Control samples were electroporated the same as AAVS1-19bbz cells except without single guide RNA (sgRNA) addition. The mean value is shown in b,c,e,g. P values were calculated by one-way ANOVA with Tukey’s multiple-comparisons test (g,h) or two-way ANOVA with Sidak’s multiple-comparisons test (f) or Tukey’s multiple-comparisons test (i). ***P < 0.001, **P < 0.01; NS, not significant.