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

Abstract

AbstractRecent 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, includingpiggyBac, have potential to obviate these shortcomings. In this study, we improved a previously developed modifiedpiggyBacsystem with superior transposition efficiency. We demonstrated that the internal domain sequences (IDS) within the 3’ terminal repeat domain of hyperactivepiggyBac(hyPB) donor vector contain dominant enhancer elements. Plasmid-free donor vector devoid of IDS was used in conjunction with a helper plasmid expressingQuantum PBase™ v2 to generate an optimalpiggyBacsystem,Quantum pBac™ (qPB), for use in T cells. Cells transfected withqPBexpressing CD20/CD19 CAR outperformed those transfected with the same donor vector and plasmid expressinghyPBtransposase in terms of CAR-T cell production. Importantly,qPByielded mainly CD8+CAR-TSCMcells, and theqPB-induced CAR-T cells effectively eliminated CD20/CD19-expressing tumor cells bothin vitroandin vivo. Our findings confirmqPBas 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.