The placebo effect Wrong patients receive treatment

Abstract

<h3>Abstract</h3> 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 <i>piggyBac</i>, have been shown to obviate these shortcomings. We have previously demonstrated the superior transposition efficiency of a modified <i>piggyBac</i> system in HEK293 cells. Here, we further advanced and broadened the therapeutic application of this modified <i>piggyBac</i> system. We demonstrated that the internal domain sequence (IDS) within the 3’ terminal repeat domain of hyperactive <i>piggyBac</i> (<i>hyPB</i>) 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 <i>Quantum pBase</i>™ v2 form the most optimal <i>piggyBac</i> system, <i>Quantum pBac</i>™ (<i>qPB</i>), in T cells. We further demonstrated that T cells transfected with <i>qPB</i> expressing CD20/CD19 CAR outperformed cells transfected with the same donor vector but with plasmid expressing <i>hyPB</i> transposase in CAR-T cell production. Importantly, we showed that <i>qPB</i> produced mainly CD8⁺ CAR-T cells that are also highly represented by T_SCM. These CAR-T cells effectively eliminated CD20/CD19-expressing tumor cells <i>in vitro</i> and in Raji-bearing immunodeficient mice. Our findings confirm that <i>qPB</i> 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. <h3>Significance Statement</h3> An effective, high capacity and safe gene integration vector system is critical to the success of gene therapy. We have previously modified a virus-free <i>piggyBac</i> transposon vector system and demonstrated its potential application in gene therapy. Here we further demonstrate that shortening of the <i>piggyBac</i> donor vector terminal repeat domain and backbone, coupled with <i>Quantum pBase</i>™ (<i>qPBase</i>) <i>v2</i>, result in the most optimal <i>Quantum pBac</i>™ (<i>qPB</i>) <i>piggyBac</i> system in human T cells. <i>qPBase v2</i> outperformed hyperactive <i>piggyBac</i> transposase in chimeric antigen receptor T (CAR-T) production. <i>qPB</i> produced CAR-T cells that are mainly CD8⁺, highly represented by T_SCM, and effectively eliminated tumors <i>in vivo</i>. Our findings solidify <i>qPB</i> as a promising virus-free vector system for therapeutic application.