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

Abstract

Recent advances in gene therapy have brought novel treatment options to multiple fields of medicine, including cancer. However, safety concerns and limited payload capacity in commonly-utilized viral vectors prevent researchers from unlocking the full potential of gene therapy. Virus-free DNA transposons, including piggyBac, have been shown to obviate these shortcomings. We have previously demonstrated the superior transposition efficiency of a modified piggyBac system in HEK293 cells. Here, we further advanced and broadened the therapeutic application of this modified piggyBac system. We demonstrated that the internal domain sequence (IDS) within the 3′ terminal repeat domain of hyperactive piggyBac (hyPB) donor vector contain dominant enhancer elements. We showed that a plasmid-free donor vector having IDS-free terminal inverted repeats in conjunction with a helper plasmid expressing Quantum pBase™ v2 form the most optimal piggyBac system, Quantum pBac™ (qPB), in T cells. We further demonstrated that T cells transfected with qPB expressing CD20/CD19 CAR outperformed cells transfected with the same donor vector but with plasmid expressing hyPB transposase in CAR-T cell production. Importantly, we showed that qPB produced mainly CD8+ CAR-T cells that are also highly represented by TSCM. These CAR-T cells effectively eliminated CD20/CD19-expressing tumor cells in vitro and in Raji-bearing immunodeficient mice. Our findings confirm that qPB is a promising virus-free vector system that is safer, and highly efficient in mediating transgene integration with the payload capacity to incorporate multiple genes.

Quantum CART(qCART), apiggyBac-basedsystem for development and production of virus-free multiplex CAR-T cell therapy

“…This study highlights the importance of keeping a low copy number of integrants per genome in gene therapy products. Our previous study has demonstrated that qPB is the most effective piggyBac-based transposon system for engineering CAR-T cells (53).…”

“…We previously showed that qPB can be advanced towards clinical application for cancer treatment with CAR-T therapy (53). However, even in the presence of artificial antigen presenting cells (aAPC), T cells from some donors still failed to expand.…”

Manufacturing CD20/CD19-targeted iCasp9 regulatable CAR-TSCMcells usingqCART, theQuantum pBac-based CAR-T system

“…Quantum pBac TM is currently the most efficient transposon for gene integration, and GF-CART01 cell therapy was our first CAR-T therapy using the novel qPB-based qCART TM system 44,45 . The qCART TM system can easily produce CAR-T cells with transgene size >7.6 kb 45 , which is not achievable by conventional lentiviral/retroviral CAR-T engineering.…”

“…14 We recently developed the most advanced version of piggyBac known as Quantum pBac TM (qPB) that is 15 times more active and potentially safer than hyperactive piggyBac in human T cells 44,45 . In our previous studies, we successfully developed a novel genetic engineering system known as Quantum CAR-T (qCART TM ) that combines efficient transgene design, evaluation, and identification of lead construct (GTailor TM ); virusfree vector (Quantum pBac TM ); effective gene delivery (Quantum Nufect TM ); and optimized cell expansion (iCellar TM ) 44,45 . We reported that qCART TM could be generically used to engineer highly potent CAR-T stem cell memory (T SCM ), a T cell population associated with enhanced anti-tumor efficacy in cancer patients.…”

“…3,13 Sleeping Beauty and piggyBac are two transposon vector systems actively being tested in CAR-T clinical trials, but they suffer from low gene transfer efficiency and limited expansion of engineered cells. 14 We recently developed the most advanced version of piggyBac known as Quantum pBac TM (qPB) that is 15 times more active and potentially safer than hyperactive piggyBac in human T cells 44,45 . In our previous studies, we successfully developed a novel genetic engineering system known as Quantum CAR-T (qCART TM ) that combines efficient transgene design, evaluation, and identification of lead construct (GTailor TM ); virusfree vector (Quantum pBac TM ); effective gene delivery (Quantum Nufect TM ); and optimized cell expansion (iCellar TM ) 44,45 .…”