Figure 4.

Confirmation of Targeted Insertion of the CAR Transgene by Digital Droplet PCR

(A) Diagram showing digital PCR strategy. Two primer pairs and probes are used: one to detect the CAR transgene inserted in TRAC and another to detect FXN and serve as a reference standard for genomic DNA. (B) Activated CD3⁺ T cells were mock-electroporated, electroporated with TRC1-2 nuclease mRNA, or mock-electroporated and transduced with 25,000 vg/cell AAV:TRAC:CAR. Digital PCR was used to quantify targeted integration of the CAR transgene in TRAC 11 days post-electroporation/transduction. (C) Activated CD3⁺ T cells were electroporated with TRC1-2 mRNA and transduced with 50,000 vg/cell AAV:TRAC:CAR. CD3⁺ and CD3⁻ groups were magnetically separated on day 8 post-transduction. Cells were stained for CD3 expression and CAR expression in the pre-separation samples and CD3 expression post-separation to confirm purity. Digital PCR was used to quantify targeted integration of the CAR transgene in TRAC in pre-separation, CD3⁺, and CD3⁻ populations.