344. Targeting piggyBac Transposition Into a User-Defined Chromosomal Locus in Human Cells

The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into the genomes of human cells. We have previously demonstrated biased PB integration into an artificial target site engineered into human cells (Kettlun et al., Molecular Therapy, 2011). We undertook the current investigation to see if we could engineer PB to target integration into a user-defined chromosomal locus in human cells. We chose to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on chromosome X. HPRT-deficient cells can be selected by growth in 6-thioguanine (6TG), so PB-mediated targeted knockout of HPRT can be used to isolate cells with targeted integration events. We used HT-1080 human fibrosarcoma cells which harbor one × chromosome for our studies. We engineered a series of 4 zinc finger proteins (ZFP) and 4 transcription activator-like (TAL) proteins fused to the PB transposase. The transposon used contained a splice-acceptor sequence followed by a beta-galactosidase-neomycin resistance fusion gene (b-Geo) which allowed measurement of gene transfer activity (measuring neomycin resistant colonies after transposition) as well as HPRT knockout efficiency (due to the splice-acceptor sequence). All HPRT-targeted ZFP-PB and TAL-PB proteins exhibited transposition activity. Chromatin immunoprecipitation assays revealed varied targeting-binding of the different PB chimeras to the HPRT locus. One ZFP-PB and one TAL-PB chimera demonstrated higher HPRT gene targeting via reproducible production of a higher number of 6TG resistant cells. Deep sequencing analysis revealed integration efficiency of 0.42% and 1% for the ZFP-PB and TAL-PB chimeric transposases in 6TG-resistant HT-1080 cells. Our study provides new insights into engineering PB to target locations in the human genome which will permit further refinements to improve targeting efficiency. Additionally, selection of cells with targeted integration events should allow isolation of “targeted-only” cells for consideration of therapeutic purposes.ss