Chimeric antigen receptor (CAR) T-cell therapy represents a promising anti-cancer immunotherapy approach. It involves genetically modifying a patient's own T-cells to create a personalized 'living anti-cancer medicine' that is capable of targeting and destroying cancer cells. It is of utmost importance to provide genomically stable CAR-T cell products, and to achieve this goal, the Sleeping Beauty (SB) transposon system is emerging as an efficient and promising gene delivery tool.

The hyperactive SB100X transposase, paired with the pT2 transposon optimised for its activity, represents an alternative approach to CAR-T cell production that is comparable in efficacy to retroviral and lentiviral delivery methods. To enhance biosafety, it is desirable to limit recombinase activity to a brief window after delivery, in order to minimise the risk of genomic toxicity. Nevertheless, the half-life of SB100X, which exceeds 30 hours, represents a considerable challenge. During this period, the transposase may potentially induce further undesired integrations or transposon remobilization. Targeting the transposase for processing by cellular degradation machinery may prove an effective solution to this issue.

To address this, the SB100X gene was modified to include an N-terminal nuclear export signal (NES) and a chaperone-mediated autophagy (CMA) signal. These modifications facilitate nuclear-to-cytoplasmic export and lysosomal degradation, resulting in a shortened half-life for the SB transposase variant.

This engineered enzyme exhibits high gene integration efficiency while demonstrating significantly increased degradation rates and nuclear export. These advancements, validated in primary T cells and various human cell lines, established an improved transposase for CAR-T cell production without compromising performance.