533. Genomic Excision of PiggyBac Transposon Cassettes by Lentiviral Protein Transduction of GagPol-Fused, Excision-Only PiggyBac Transposase

Abstract

The PiggyBac (PB) transposon system is a potent nonviral gene delivery tool with relevance in both gene therapy and cell reprogramming for production of induced pluripotent stem cells (iPSCs). Moreover, by taking advantage of the ability of the PB transposase to excise transposon-embedded gene cassettes from the genome without leaving footprints, the PB system is unique in facilitating seamless genome editing. The need for intracellular production of the transposase, however, raises concerns related to delivery and to cytotoxicity caused by sustained transposase expression and insertional mutagenesis. Furthermore, transposon re-integration may decrease the overall efficiency of the PB-mediated excision as well as increasing the risk of adverse secondary insertions. Based on our previous work, we present a new approach for lentivirus-based delivery of PB transposase. By fusing the hyperactive PB transposase, hyPBase, to the C-terminus of the GagPol polyprotein, we show robust incorporation and subsequent release of the transposase in matured lentiviral particles. Furthermore, in an effort to limit transposon re-integration, we engineered a hyPBase variant carrying three missense mutations. This novel hyPBase variant, hyPBaseExc+/Int−, demonstrates integration levels very close to background levels, thus limiting the risk of reintegration. Notably, the ability of hyPBaseExc+/Int− to excise transposons from plasmid-borne as well as from genomically integrated PB transposon cassettes is increased up to 6.3-fold relative to the original hyPBase transposase. By fusing the hyPBaseExc+/Int− to the C-terminus of GagPol, transposase protein can be efficiently delivered to cells by lentiviral protein transduction and, in our model system, performs seamless genomic excision of PB transposon cassettes in a copy number and dose-dependent manner, resulting in excision in up to 23.6% of virus-treated cells. Using a transposon containing the puro-deltaTK transgene cassette, we furthermore show that cells with successful excision can be enriched 17-fold by negative selection with FIAU. We believe that protein transduction of hyPBaseExc+/Int− may increase the applicability and safety of transposase-directed genomic excision in iPSCs and in hard-to-transfect cell types including hematopoietic cells.