Abstract
Transposons, as non-viral vectors, provide an efficient and secure method for stable gene delivery and have been successfully applied in human gene therapies. The engineering of transposase has significantly improved the efficiency of various transposon systems, including chimeric antigen receptor (CAR)-T cell engineering. In this study, multiple engineering strategies were implemented to enhance the efficiency and safety of the Baize (BZ) transposon system, which was derived from the ZB (the wild-type BZ, BZwt) and has been proven to be an effective tool for genetic manipulation in vertebrates. Through designed engineering and combinatorial mutagenesis in vitro, several hyperactive BZ transposase variants with higher transposition activity, cargo capacity, and integration safety were developed. At optimal activity levels, BZ325 surpassed BZwt by ∼1.2-fold and 2.3-fold at 500 ng and 10 ng dosages of donor plasmids, respectively. Furthermore, reducing the size of BZ donor vector backbone significantly increased CAR-T modification efficiency without compromising its function. Notably, BZ325, BZ326, and especially BZ327 exhibited significantly higher CAR-T engineering rates and CAR expression levels than BZwt. Overall, the engineering of the BZ transposon system significantly enhanced its transposition activity, cargo capacity, and safety, providing a compelling tool for gene transfer applications and emphasizing its potential in gene therapy.