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

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 limited payload capacity of commonly utilized viral vectors. Virus-free DNA transposons, including piggyBac, have the potential to obviate these shortcomings. In this study, we improved a previously 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. qPB outperformed hyPB in CD20/CD19 CAR-T production in terms of performance as well as yield of the CAR-T cells produced. Furthermore, qPB also produced CAR-T cells with lower donor-associated variabilities compared to lentiviral vector. Importantly, qPB yielded mainly CD8+ CAR-TSCM cells, and the qPB-produced 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 an enhanced payload capacity to incorporate multiple genes. This highly efficient and potentially safe system will be expected to further advance gene therapy applications.

Region-specific epithelial cell dynamics during branching morphogenesis.

BACKGROUND: Epithelial cells of developing embryonic organs, such as salivary glands, can display substantial motility during branching morphogenesis. Their dynamic movements and molecules involved in their migration are not fully characterized. RESULTS: We generated transgenic mice expressing photo-convertible KikGR and tracked the movements of individual cells highlighted by red fluorescence in different regions of developing salivary glands. Motility was highest for outer bud epithelial cells adjacent to the basement membrane, lower in inner bud cells, and lowest in duct cells. The highly motile outer cells contacting the basement membrane were pleomorphic, whereas inner cells were rounded. Peripheral cell motility was disrupted by antibodies inhibiting α6+ß1 integrins and the nonmuscle myosin II inhibitor blebbistatin. Inner bud cell migration was unaffected by these inhibitors, but their rate of migration was stimulated by inhibiting E-cadherin. CONCLUSIONS: Cell motility in developing salivary glands was highest in cells in contact with the basement membrane. The basement membrane-associated motility of these outer bud cells depended on integrins and myosin II, but not E-cadherin. In contrast, motility of inner bud cells was restrained by E-cadherin. These findings identify the importance of integrin-dependent basement membrane association for the morphology, tissue organization, and lateral motility of morphogenetic epithelial cells.