Genome-wide target profiling of piggyBac and Tol2in HEK 293: pros and cons for gene discovery and gene therapy

Abstract

BackgroundDNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side-by-side comparison of the two transposon systems in the same setting to evaluate their advantages and disadvantages for use in gene therapy and gene discovery.ResultsWe have observed that (1) the Tol2 transposase (but not piggyBac) is highly sensitive to molecular engineering; (2) the piggyBac donor with only the 40 bp 3'-and 67 bp 5'-terminal repeat domain is sufficient for effective transposition; and (3) a small amount of piggyBac transposases results in robust transposition suggesting the piggyBac transpospase is highly active. Performing genome-wide target profiling on data sets obtained by retrieving chromosomal targeting sequences from individual clones, we have identified several piggyBac and Tol2 hotspots and observed that (4) piggyBac and Tol2 display a clear difference in targeting preferences in the human genome. Finally, we have observed that (5) only sites with a particular sequence context can be targeted by either piggyBac or Tol2.ConclusionsThe non-overlapping targeting preference of piggyBac and Tol2 makes them complementary research tools for manipulating mammalian genomes. PiggyBac is the most promising transposon-based vector system for achieving site-specific targeting of therapeutic genes due to the flexibility of its transposase for being molecularly engineered. Insights from this study will provide a basis for engineering piggyBac transposases to achieve site-specific therapeutic gene targeting.

Hyperactive piggyBac Gene Transfer in Human Cells and In Vivo

“…As piggyBac is capable of catalyzing transposition in a range of clinically relevant human cell types Woltjen et al, 2009;Chen et al, 2010), the use of a hyperactive transposase should enable more efficient production of sufficient numbers of cells for therapies. Also, hyperactive piggyBac maintained full activity with the addition of a 24-kDa DNA-binding domain, whereas SB100X was reduced in activity by approximately 50%, consistent with previously described results of adding a tag to piggyBac (Wilson et al, 2007;Meir et al, 2011) and earlier versions of Sleeping Beauty (Wilson et al, 2005;Ivics et al, 2007;Yant et al, 2007). The ability to be modified without loss of activity increases potential applications of the piggyBac transposon system, such as targeting the transposase to specific genomic locations by the addition of a site-specific DNA-binding domain, as we have recently described (Kettlun et al, 2011).…”

“…In cell therapy applications, hyperactive piggyBac should permit the generation of more cells stably expressing transgenes. In addition, recent studies have described alterations of the piggyBac inverted repeat elements to increase transposition, but these have not yet found their way into common use (Lacoste et al, 2009;Meir et al, 2011). The use of these elements with the hyperactive transposase could increase gene delivery to levels greater than we have observed in our experiments.…”

“…A number of previous reports have examined the integration preferences of piggyBac in a variety of cell types (Wilson et al, 2007;Galvan et al, 2009;Huang et al, 2010;Meir et al, 2011). Our previous results have indicated that native piggyBac is more prone to integrate in or near genes than Sleeping Beauty, but is significantly less likely than retroviral vectors to integrate in or near proto-oncogenes.…”

“…specific sites in the genome (Wilson et al, 2005;Kettlun et al, 2011). We and others have previously reported that adding a tag or protein domain to the Sleeping Beauty transposase significantly attenuates transposition (Wilson et al, 2005;Ivics et al, 2007;Yant et al, 2007), whereas doing the same to piggyBac has no apparent effect on its activity (Wilson et al, 2007;Meir et al, 2011); however, the effect of a tag on the activity of the 7pB and SB100X transposases has not been demonstrated.…”

Excision Efficiency Is Not Strongly Coupled to Transgenic Rate: Cell Type-Dependent Transposition Efficiency ofSleeping BeautyandpiggyBacDNA Transposons

“…All have high transgenic potential and can mobilize large cargos (Rostovskaya et al, 2012;Wang et al, 2014), which is a considerable factor as compared with viral packaging systems. For safety considerations in gene therapy, SB seems to be the most ideal system because of its favorable integration profile: there is no obvious preference at the genomic level, and therefore the delivered transgene is integrated randomly, lowering the risk of undesirable insertional mutagenesis (Galvan et al, 2009;Grabundzija et al, 2010;Izsvák et al, 2010;Meir et al, 2011;Burnight et al, 2012;M.A. Li et al, 2013).…”