387. Advantageous Properties of the piggyBac Transposon System for Gene Transfer in Human Cells

Transposon systems permit non-viral delivery of genetic cargo into the genome of human cells and hold promise for eventual use in gene therapy. Transposon systems with activity in vertebrates include Sleeping Beauty (SB) and Mos1 of the Tc1/Mariner family and piggyBac which represents a different class of transposable element. We undertook evaluation of these three different transposable elements in human cells in order to compare the properties between each transposon system. This permitted direct comparison of the activity of native and hyperactive SB, native and hyperactive Mos1, and native piggyBacin plasmid vectors differing only by the transposase cDNA and inverted repeat (IR) elements. Mos1 required full internal sequences to be present for measurable transposition activity in human cells. The piggyBac transposable element could be reduced to a 311bp left IR and a 236bp right IR with retention of full activity. Evaluation of overall transposable activity revealed Mos1 << SB < piggyBac in cultured human cells. Hyperactive mutants of the Mos1 transposase were evaluated and showed little increase in transposition over native Mos1 in human cells. Interestingly, piggyBac showed more activity than native or even hyperactive SB (two-to-ten fold more active than hyperactive SB12 transposase combined with hyperactive pT3 transposon). Additionally, overproduction inhibition, a known major limitation of SB, was lacking with piggyBac. We evaluated piggyBac transposition following transient transfection of 50ng, 100ng, and 2μg transposon DNA while varying the transposase DNA amount. In all cases, piggyBac lacked overproduction inhibition while SB transposition could be driven to very low levels at higher amounts of transposase DNA. Such results revealed that piggyBac transposition was limited only by the amount of transposon DNA present. Finally, we evaluated the addition of a zinc finger DNA binding domain element to the N-terminus of the different transposase enzymes. Zinc finger DNA binding domain addition ablated Mos1 activity and decreased SB activity which was only reliably measured using a combination of hyperactive transposase and transposon mutants. However, chimeric-piggyBac transposase activity appeared unaffected by N-terminal zinc finger DNA binding domain addition when compared to the native system. These results demonstrate that the piggyBac transposon system has advantageous properties when compared to other transposon systems with activity in human cells thereby making it a viable alternative system for gene therapy.