Figure 3.

Combining TRC1-2 and an AAV Donor Template Results in Highly Efficient HDR-Mediated Insertion of an Anti-CD19 CAR into the TRAC Locus and Simultaneous Disruption of TCR Expression

(A) Diagram of the AAV vector used to transduce cells. AAV:TRAC:CAR contains a CD19 CAR transgene with expression driven by the JeT promoter and flanked by homology arms on the 5′ (L TRAC HA) and 3′ (R TRAC HA) sides to enable targeted integration. (B) Diagram of the PCR used to confirm CAR integration by amplification with one primer located within the CAR and one primer in TRAC outside of the homology arms at both the 5′ and 3′ ends to generate 1,872-bp and 1107-bp products, respectively. (C and D) T cells were mock-electroporated or electroporated with TRC1-2 mRNA and then immediately transduced with the indicated amounts of AAV:TRAC:CAR. (C) PCR was used to confirm the presence of the CAR transgene integrated in the TRAC locus on day 3 post-electroporation and transduction as outlined in (B). (D) CAR and CD3 expression were evaluated by flow cytometry on days 3 and 8 post-electroporation and transduction.