Base editing can be used to create a multiplexed edited CART modified at multiple loci. (A) NGS (left) and flow cytometry expression (right) of 4 proteins knocked out from the CART 7CAR8 using CBE. CD52, CD7, PDCD1, and TRAC were all knocked out with high efficiency. (B) Protein reduction measured using flow cytometry for the 4 editing targets. (C) Translocation analysis using the UDiTaS assay between the 4 on-target editing sites for EP-only control cells, CBE-edited cells, and spCas9-edited cells produced from 3 healthy donors. (D) Expression of CAR measured in CD8⁻ and CD8+ T cells using flow cytometry in 7CAR8 and untransduced T cells. X-axis depicts CAR⁺ expression measured by staining with fluorophore-conjugated recombinant human CD7 protein. Y-axis depicts CD8 expression. (E) 7CAR8 cells produced from 3 separate donors cocultured with GFP-luciferase expressing T-ALL cell line CCRF-CEM in a 4:1, 2:1, and 1:1 E:T ratio. GFP expression over time was measured using the IncuCyte system. (F) Mice injected with the cell line CCRF-CEM modified to expressed GFP-luciferase were randomized to be treated with vehicle, untransduced T cells, or increasing doses of 7CAR8 (n = 10 mice per arm). A dose-dependent decrease in the surrogate disease marker total flux over time was seen in mice treated with 7CAR8. (G) Representative images of mice treated with vehicle and different doses of 7CAR8, with mice receiving the highest dose of 7CAR8 having no disease present.