Endogenous Transposase Source in Human Cells Mobilizes piggyBac Transposons
Authors: Zoltán Ivics
Journal: Molecular Therapy (2016)

piggyBac (PB) transposons are mobile genetic elements widespread in nature. One particular PB transposon originally isolated from insects has been developed as a nonviral gene transfer tool, with human gene therapy at the far end of potential applications. Transposons can occasionally be recruited for useful functions by their hosts, and the human genome has five such "domesticated" genes derived from PB transposases. In a recent study, Henssen et al.1Henssen AG Henaff E Jiang E Eisenberg AR Carson JR Villasante CM et al.Genomic DNA transposition induced by human PGBD5.Elife. 2015; 4: 10565Crossref Scopus (46) Google Scholar examined one such gene product, PGBD5 (piggyBac-derived 5), with the goal of probing the potential of this protein to catalyze bona fide transposition. Very unexpectedly, the study shows that PGBD5 can mobilize the insect PB transposon in human cell culture. Thus, despite the vast evolutionary distance between PGBD5 and insect PB transposons, there appears to be a cross-reaction between a catalytically active endogenous human transposase and transposon vector sequences that are exogenously delivered into human cells. The findings may have implications for genomic stability of PB vector insertions in human applications. Many transposons, including members of the PB superfamily,2Yusa K piggyBac Transposon.Microbiol Spectr 3: MDNA3-0028-2014. 2015; Google Scholar transpose via a "cut-and-paste" reaction involving binding of the transposase to the ends of an element defined by inverted terminal repeats (ITRs), excise the transposon from its original DNA context, and insert the excised element into a new DNA sequence (Figure 1a). The founding member of the PB superfamily was discovered as an insertion in a baculovirus (hence the name) in a cell line isolated from the cabbage looper moth Trichoplusia ni.3Fraser MJ Brusca JS Smith GE Summers MD Transposon-mediated mutagenesis of a baculovirus.Virology. 1985; 145: 356-361Crossref PubMed Scopus (105) Google Scholar Subsequent studies revealed that members of the PB superfamily are widespread in nature and can be found in fungi, plants, insects, crustaceans, urochordates, amphibians, fishes, and mammals.4Sarkar A Sim C Hong YS Hogan JR Fraser MJ Robertson HM et al.Molecular evolutionary analysis of the widespread piggyBac transposon family and related "domesticated" sequences.Mol Genet Genomics. 2003; 270: 173-180Crossref PubMed Scopus (134) Google Scholar The T. ni PB transposon has 13-bp ITRs at its ends, inserts into TTAA target sequences, and is transpositionally active in a wide range of cell types and species. Thus, the PB transposon has become a widely used, popular tool for a variety of applications including animal transgenesis,5Ding S Wu X Li G Zhuang Y Xu T Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice.Cell. 2005; 122: 473-483Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar mutagenesis screens to functionally annotate genes in vitro6Pettitt SJ Tan EP Yusa K piggyBac transposon-based insertional mutagenesis in mouse haploid embryonic stem cells.Methods Mol Biol. 2015; 1239: 15-28Crossref PubMed Scopus (10) Google Scholar,7Li MA Pettitt SJ Yusa K Bradley A Genome-wide forward genetic screens in mouse ES cells.Methods Enzymol. 2010; 477: 217-242Crossref PubMed Scopus (18) Google Scholar and in vivo,8Rad R Rad L Wang W Cadinanos J Vassiliou G Rice S et al.PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice.Science. 2010; 330: 1104-1107Crossref PubMed Scopus (179) Google Scholar,9Rad R Rad L Wang W Strong A Ponstingl H Bronner IF et al.A conditional piggyBac transposition system for genetic screening in mice identifies oncogenic networks in pancreatic cancer.Nat Genet. 2015; 47: 47-56Crossref PubMed Scopus (55) Google Scholar induced pluripotent stem cell reprogramming,10Woltjen K Michael IP Mohseni P Desai R Mileikovsky M Hämäläinen R et al.). piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.Nature. 2009; 458: 766-770Crossref PubMed Scopus (1444) Google Scholar,11Yusa K Rad R Takeda J Bradley A Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon.Nat Methods. 2009; 6: 363-369Crossref PubMed Scopus (475) Google Scholar and gene therapy.12Di Matteo M Samara-Kuko E Ward NJ Waddington SN McVey JH Chuah MK et al.Hyperactive piggyBac transposons for sustained and robust liver-targeted gene therapy.Mol Ther. 2014; 22: 1614-1624Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar,13Loperfido M Jarmin S Dastidar S Di Matteo M Perini I Moore M et al.piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.Nucleic Acids Res. 2016; 44: 744-760Crossref PubMed Scopus (22) Google Scholar,14Cooney AL Singh BK Sinn PL Hybrid nonviral/viral vector systems for improved piggyBac DNA transposon in vivo delivery.Mol Ther. 2015; 23: 667-674Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar,15Saha S Nakazawa Y Huye LE Doherty JE Galvan DL Rooney CM et al.piggyBac transposon system modification of primary human T cells.J Vis Exp. 2012; : e4235PubMed Google Scholar,16Manuri PV Wilson MH Maiti SN Mi T Singh H Olivares S et al.piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies.Hum Gene Ther. 2010; 21: 427-437Crossref PubMed Scopus (111) Google Scholar Transposons are not only useful tools for genetic engineering, but have also played a key role in the evolution of novel gene functions in nature. Indeed, it is now widely recognized that, through an evolutionary process termed co-option, transposon-derived DNA, RNA, and protein components have been recurrently recruited into cellular pathways as regulatory elements.17Feschotte C Transposable elements and the evolution of regulatory networks.Nat Rev Genet. 2008; 9: 397-405Crossref PubMed Scopus (889) Google Scholar Specifically, several transposases have been "domesticated" as functional host proteins probably by virtue of their ability to bind, loop, and, occasionally, recombine transposon-derived sites throughout the genome. V(D)J recombination, a site-specific recombination reaction in the immune system of jawed vertebrates, is incontestably the most spectacular example for a domesticated transposition reaction that can derive complex and crucial functions in the host. In this process, which occurs during lymphocyte development, V (variable), D (diversity), and J (joining) gene segments are rearranged to generate a large repertoire of T-cell surface receptor and immunoglobulin molecules necessary for the recognition of diverse pathogens (reviewed in ref. 18Jones JM Gellert M The taming of a transposon: V(D)J recombination and the immune system.Immunol Rev. 2004; 200: 233-248Crossref PubMed Scopus (80) Google Scholar). The RAG1/RAG2 V(D)J recombinase is a domesticated transposase that evolved out of a Transib family transposon in a common ancestor of jawed vertebrates.19Kapitonov VV Jurka J RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons.PLoS Biol. 2005; 3: e181Crossref PubMed Scopus (360) Google Scholar Genome sequences of various species have also revealed a number of previously unrecognized genes that were derived from various transposable elements including PB transposases.4Sarkar A Sim C Hong YS Hogan JR Fraser MJ Robertson HM et al.Molecular evolutionary analysis of the widespread piggyBac transposon family and related "domesticated" sequences.Mol Genet Genomics. 2003; 270: 173-180Crossref PubMed Scopus (134) Google Scholar,20Lander ES Linton LM Birren B Nusbaum C Zody MC Baldwin J et al.International Human Genome Sequencing Consortium (2001). Initial sequencing and analysis of the human genome.Nature. 2001; 409: 860-921Crossref PubMed Scopus (17711) Google Scholar For example, the human genome contains five genes that were apparently derived from PB transposases, PGBD1–5. PGBD5 can be found in all vertebrates including lampreys as well as in lancelets, indicating that this gene was domesticated more than 500 million years ago in a common ancestor of cephalochordates and vertebrates.21Pavelitz T Gray LT Padilla SL Bailey AD Weiner AM PGBD5: a neural-specific intron-containing piggyBac transposase domesticated over 500 million years ago and conserved from cephalochordates to humans.Mob DNA. 2013; 4: 23Crossref PubMed Scopus (18) Google Scholar PGBD5 encodes a putative full-length transposase that is preferentially expressed in the early embryo and in the adult brain and central nervous system in vertebrates, including humans.21Pavelitz T Gray LT Padilla SL Bailey AD Weiner AM PGBD5: a neural-specific intron-containing piggyBac transposase domesticated over 500 million years ago and conserved from cephalochordates to humans.Mob DNA. 2013; 4: 23Crossref PubMed Scopus (18) Google Scholar Henssen et al.1Henssen AG Henaff E Jiang E Eisenberg AR Carson JR Villasante CM et al.Genomic DNA transposition induced by human PGBD5.Elife. 2015; 4: 10565Crossref Scopus (46) Google Scholar set out to investigate whether PGBD5 is transpositionally active. They transfected human HEK293 cells with a transposon donor plasmid containing a neomycin resistance gene (neo)-tagged T. ni PB transposon and a helper plasmid expressing PGBD5. Clonogenic assays indicated active transposition of the neo-tagged transposons from the plasmids into the cellular genome; the reaction was apparently dependent on full-length PGBD5 and the presence of PB ITRs flanking neo, indicating a bona fide transpositional reaction. Further molecular tests revealed reconstitution of TTAA sequences at the donor sites (a characteristic feature of PB excision22Mitra R Fain-Thornton J Craig NL piggyBac can bypass DNA synthesis during cut and paste transposition.EMBO J. 2008; 27: 1097-1109Crossref PubMed Scopus (158) Google Scholar), as well as precise genomic integration events into TTAA target sequences in the human genome. Evolutionary preservation suggests a biological relevance of the transpositional activity of PGBD5. Although the physiologically relevant, endogenous sites on which PGBD5 can potentially act are currently unknown, the human genome contains thousands of transposon-like sequences with sequence similarity to the ITRs of the T. ni PB transposon.1Henssen AG Henaff E Jiang E Eisenberg AR Carson JR Villasante CM et al.Genomic DNA transposition induced by human PGBD5.Elife. 2015; 4: 10565Crossref Scopus (46) Google Scholar Thus, PGBD5 might be involved in a DNA recombination reaction involving such sites. On the basis of the expression pattern of PGBD5, it is tempting to speculate that, as with LINE-1 retrotransposition in the brain,23Muotri AR Chu VT Marchetto MC Deng W Moran JV Gage FH Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition.Nature. 2005; 435: 903-910Crossref PubMed Scopus (691) Google Scholar PGBD5-mediated DNA recombination may contribute to somatic mosaicism in the central nervous system. Regardless of the biological function of PGBD5, the ability of this protein to mediate canonical "cut-and-paste" transposition of T. ni PB transposons in human cells is astonishing given the divergence of PGBD5 and insect PB transposons. However, cross-reaction of transposases and divergent transposon sequences is not without precedent. For example, THAP9, a domesticated P-element transposase in humans, can mobilize P elements from Drosophila melanogaster in human cells.24Majumdar S Singh A Rio DC The human THAP9 gene encodes an active P-element DNA transposase.Science. 2013; 339: 446-448Crossref PubMed Scopus (39) Google Scholar Thus, the specificity of certain transpositional reactions might be significantly relaxed. Relaxed specificity of the PGBD5 transposase is a potential issue in the context of genetic engineering of human cells with PB transposon vectors. Indeed, a potential concern with the use of the PB transposon system has been25Feschotte C The piggyBac transposon holds promise for human gene therapy.Proc Natl Acad Sci USA. 2006; 103: 14981-14982Crossref PubMed Scopus (38) Google Scholar,26VandenDriessche T Ivics Z Izsvak Z Chuah MK Emerging potential of transposons for gene therapy and generation of induced pluripotent stem cells.Blood. 2009; 114: 1461-1468Crossref PubMed Scopus (119) Google Scholar that the PB superfamily is well represented in the human genome, and thus components of the vector system might interact with endogenous human factors in unpredictable ways, thereby compromising genomic integrity. First, there are ∼2,000 PB-like elements dispersed throughout the human genome, with the MER85 and MER75 families being among the most recently amplified DNA transposon families recognizable in human DNA,1Henssen AG Henaff E Jiang E Eisenberg AR Carson JR Villasante CM et al.Genomic DNA transposition induced by human PGBD5.Elife. 2015; 4: 10565Crossref Scopus (46) Google Scholar,4Sarkar A Sim C Hong YS Hogan JR Fraser MJ Robertson HM et al.Molecular evolutionary analysis of the widespread piggyBac transposon family and related "domesticated" sequences.Mol Genet Genomics. 2003; 270: 173-180Crossref PubMed Scopus (134) Google Scholar,25Feschotte C The piggyBac transposon holds promise for human gene therapy.Proc Natl Acad Sci USA. 2006; 103: 14981-14982Crossref PubMed Scopus (38) Google Scholar and a large fraction of these PB-like transposons have probably preserved the ITR sequences required for transposition. Thus, an exogenously supplied T. ni PB transposase could mobilize some of the human PB-like transposons. This possibility was experimentally addressed by Saha et al.,27Saha S Woodard LE Charron EM Welch RC Rooney CM Wilson MH Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells.Nucleic Acids Res. 2015; 43: 1770-1782Crossref PubMed Scopus (34) Google Scholar who found that expression of the insect PB transposase did not mobilize endogenous, human PB-like sequences, and did not lead to an increase in DNA double-strand breaks in the human genome. Second, the endogenous, domesticated PGBD1–5 proteins could excise and/or remobilize a transgene flanked by the ITRs of a PB vector (Figure 1b). This would pose a problem, because transgenes delivered by any vector system should ideally behave as stable constituents of target genomes. That is, once integrated, a therapeutic transposon should not move again. Indeed, one key feature of transposon-based gene transfer systems is conditionality of the transposition reaction; that is, genomic transgene integration should only occur while the transposase is exogenously and transiently supplied28Ivics Z Li MA Mátés L Boeke JD Nagy A Bradley A et al.Transposon-mediated genome manipulation in vertebrates.Nat Methods. 2009; 6: 415-422Crossref PubMed Scopus (242) Google Scholar (Figure 1a). The Henssen et al. study1Henssen AG Henaff E Jiang E Eisenberg AR Carson JR Villasante CM et al.Genomic DNA transposition induced by human PGBD5.Elife. 2015; 4: 10565Crossref Scopus (46) Google Scholar delivers strong evidence that human PGBD5 can mobilize an insect PB transposon, suggesting the possibility of ongoing remobilization of genomically integrated PB transgenes (Figure 1b). There are at least two important considerations when evaluating this scenario. First, Henssen et al. used ectopic overexpression of PGBD5 together with transiently transfected, episomal PB transposon donor plasmids. It is currently unknown whether endogenous levels of PGBD5 are sufficient for PB mobilization and whether chromosomally located PB transposons can "re-hop" in the presence of PGBD5. Further investigations are required to clarify these questions. Second, as noted before, PGBD5 has a fairly restricted pattern of expression in human tissues.21Pavelitz T Gray LT Padilla SL Bailey AD Weiner AM PGBD5: a neural-specific intron-containing piggyBac transposase domesticated over 500 million years ago and conserved from cephalochordates to humans.Mob DNA. 2013; 4: 23Crossref PubMed Scopus (18) Google Scholar Screening publicly available gene expression databases (microarray data) indeed reveals relatively low expression of PGBD5 in fibroblasts, mesenchymal stem cells, smooth muscle cells, CD34+ hematopoietic stem cells, and T and B lymphocytes (Figure 1c). Consistent with chromatin remodeling of the PGBD5 promoter in human embryonic stem cells,21Pavelitz T Gray LT Padilla SL Bailey AD Weiner AM PGBD5: a neural-specific intron-containing piggyBac transposase domesticated over 500 million years ago and conserved from cephalochordates to humans.Mob DNA. 2013; 4: 23Crossref PubMed Scopus (18) Google Scholar PGBD5 shows robust expression in pluripotent stem cells, including ES and induced pluripotent stem cells (Figure 1c), and relatively high expression in the neural lineage upon in vitro differentiation (Figure 1c). In addition, we find a previously unnoted, significant expression of PGBD5 in hepatocytes and keratinocytes (Figure 1c). Thus, a potential safety issue posed by possible PB transposon vector instability must be critically addressed in cell types that express PGBD5 in the context of clinical applications.