Fig. 4. Programmable human cell delivery generates gene-edited CAR T cells in vivo.

a, Summary of T cell-targeted Cas9-EDVs and lentivirus tested in PBMC-humanized mice. Both particles display multiplexed scFvs (CD3 scFv-3, CD4 scFv-1 and CD28 scFv-2). The Cas9-EDV vector co-packages a lentiviral-encoded CAR-2A-mCherry transgene and Cas9 RNP complexes to disrupt the TRAC gene; the lentivirus encodes the CAR-2A-mCherry transgene. b, Experimental schematic for testing T cell-targeted Cas9-EDVs and lentivirus in PBMC-humanized mice by intravenous (I.V.) retro-orbital injections. c, Representative flow cytometry plots demonstrating that CAR-expressing human T cells are detectable in the spleens of PBMC-humanized mice 10 days post administration of 1.5 × 10⁹ Cas9-EDV (n = 2 animals) or lentivirus (n = 3 animals) but not in mice administered PBS (n = 3), quantified in d. e,f, Gene editing is observed in CAR⁻ and CAR⁺ human T cells isolated from mice treated with T cell-targeted Cas9-EDVs (n = 2 animals) and T cell-targeted lentivirus (n = 3 animals). One CAR⁺ lentivirus sample was excluded in f because of failing sequencing. g, CAR-expressing human T cells are detectable in the spleens of PBMC-humanized mice 10 days post administration of 6.2 × 10⁸ Cas9-EDV (n = 8 animals) or lentivirus (n = 8 animals) but not in mice administered PBS (n = 4 animals). P values calculated by means of Dunnett’s multiple comparison test after Brown–Forsythe and Welsh one-way ANOVA. h,i, Genome editing is observed in CAR⁻ and CAR⁺ human T cells isolated from mice treated with T cell-targeted Cas9-EDVs (n = 8 animals per group). Significance calculated by two-sided unpaired t-test. Comparison in i is not significant (P > 0.05). For all plots, black lines indicate the median of the data set. LOD, limit of detection as defined by the average modified reads from lentiviral-treated samples.