120. Generation of Transgene-Only Gene-Modified Human Cells Via Transposition
Authors: Lauren E. Woodard, Sunandan Saha, Elizabeth M. Charron, Richard C. Welch, Matthew H. Wilson
Journal: Molecular Therapy (2015)

DNA transposons such as piggyBac and Sleeping Beauty employ a cut-and-paste mechanism of transposition that has proven effective for numerous gene transfer and cellular reprogramming applications. Therapeutic transposons are carried into cells usually on plasmid DNA vectors either on the same plasmid as the transposase expression cassette in "cis" or on another plasmid in "trans." We examined the fate of the remaining plasmid backbone after transposition. It has been assumed that the recircularized plasmid backbone remains episomal and is quickly lost from dividing cells. We delivered transposons containing a neomycin resistance gene and the transposase in either "cis" or "trans" to human embryonic kidney (HEK-293) cells, then selected the cells for 8 weeks and evaluated transposase expression by immunoblot. All of the piggyBac and 5 out of 6 of the Sleeping Beauty "cis" transfections resulted in transposase-positive clones. In contrast, none of the piggyBac "trans" transfections resulted in transposase-positive clones. We were concerned that the prolonged expression of the transposase might result in some instability of the transgenes delivered "cis" in vivo; however, we found that mice that received a piggyBac "cis" luciferase transposon vector by hydrodynamic injection had long-term luciferase values that were not statistically different from mice receiving the plasmids in "trans." We next evaluated if piggyBac "trans" delivery could be modified to reduce the amount of backbone integration and found that clones that were transfected with lower amounts of transposase showed a decrease in the frequency of backbone integrations. We found no evidence that transposition increases the frequency of integration of co-delivered linear DNA. We sequenced backbone integration events and found that that the majority of the time, once the transposon is excised from its parent plasmid, the plasmid backbone is repaired by re-ligation and integrated into the genome at a site other than the excision site. Alternatively, a rare event may occur in which the linearized plasmid backbone can be integrated without excision repair. None of the integration events occurred at the TTAA tetranucleotide sequence, indicating that they are not directly mediated by piggyBac transposase. We next devised a strategy to measure and eliminate the backbones from human cells. We created a plasmid containing the neomycin-resistance gene in the transposon portion of the plasmid and the eGFP marker in the backbone. Transfected HEK-293 cells were selected with geneticin and sorted for eGFP. While the selected cells contained approximately ten copies of the transposon per diploid cell, unsorted cells typically had one to two copies of plasmid backbone per diploid genome. eGFP sorting effectively eliminated recovery of backbone-negative transgene-only cells. We expect further improvements to improve recovery of transgene-only cells. We will also investigate the extent of backbone integration in therapeutically relevant cell types and in vivo.