Bryce Wick

CAR-T cells have shown promise in the clinic in the treatment of blood cancers. Most often, these are created using viral vectors. Viral vectors have size limitations, the risk of insertional mutagenesis, and are costly to produce. Therefore, an attractive alternative is transposons, which are less expensive and safer than viral delivery methods. Here, we create CAR-T cells using the TC Buster transposase system and achieve an almost pure population of CAR+ T cells using drug selection. Briefly, primary human T cells were stimulated with Dynabeads, electroporated two days later, and restimulated. The cargo was a transposon containing a CD19 CAR, a mutant DHFR gene for drug selections with methotrexate (MTX), and GFP for integration analysis via flow cytometry, and the transposase was either a standard TCB transposase or a hyperactive one. After a week of drug selection, flow cytometry revealed that either transposase was able to produce a >99% GFP+ population. Without drug selection, the standard transposase produced a ~14% GFP+ population compared to the hyperactive transposases’ ~58% GFP+ population. In a killing assay, these cells were able to kill almost all CD19+ Raji-Luc cells plated at an E:T ratio of 3:1. This work shows that using the TC Buster transposase system allows for effective engineering of CAR-T cells and that drug selection can create an almost pure CAR+ population.