Quantum pBac: An effective, high-capacity piggyBac-based gene integration vector system for unlocking gene therapy potential

Abstract

Recent advances in gene therapy have brought novel treatment options for cancer. However, the full potential of this approach has yet to be unlocked due to the safety concerns and limited payload capacity of commonly utilized viral vectors. Virus-free DNA transposons, including piggyBac, have potential to obviate these shortcomings. In this study, we improved a previously developed modified piggyBac system with superior transposition efficiency. We demonstrated that the internal domain sequences (IDS) within the 3 terminal repeat domain of hyperactive piggyBac (hyPB) donor vector contain dominant enhancer elements. Plasmid-free donor vector devoid of IDS was used in conjunction with a helper plasmid expressing Quantum PBase v2 to generate an optimal piggyBac system, Quantum pBac (qPB), for use in T cells. Cells transfected with qPB expressing CD20/CD19 CAR outperformed those transfected with the same donor vector and plasmid expressing hyPB transposase in terms of CAR-T cell production. Importantly, qPB yielded mainly CD8+ CAR-TSCM cells, and the qPB- induced CAR-T cells effectively eliminated CD20/CD19-expressing tumor cells both in vitro and in vivo. Our findings confirm qPB as a promising virus-free vector system with a payload capacity to incorporate multiple genes. This system is highly efficient and potentially safe for mediating transgene integration.