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 T cell receptor alpha constant (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 I.V. (intravenous) 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 1.5x10⁹ 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-negative and CAR-positive 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-positive lentivirus sample was excluded in (f) due to failing sequencing. g, CAR-expressing human T cells are detectable in the spleens of PBMC-humanized mice 10 days post administration of 6.2x10⁸ 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-negative and CAR-positive 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. PBS = phosphate-buffered saline.