Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis

OPEN SUBJECT AREAS: GENETIC VECTORS MOBILE ELEMENTS Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis Syandan Chakraborty1, HaYeun Ji1, Jack Chen1, Charles A. Gersbach1,2,3 & Kam W. Leong1 Received 12 August 2014 Accepted 19 November 2014 Published 10 December 2014 Correspondence and requests for materials should be addressed to K.W.L. (kam.leong@ duke.edu.) 1 Department of Biomedical Engineering, Duke University, Durham, North Carolina, 27708, USA, 2Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, 27708, USA, 3Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, 27708, USA. Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed ‘‘cut-and-paste’’ mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In this study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the separation of the transposase element from the polyA sequence after transposition leads to its deactivation. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells. T ransgenesis plays a crucial role in unraveling the function of various genes in developmental processes and disease states1,2, recombinant protein generation3,4, gene therapy5,6 and reprogramming of somatic cells7,8. Viral vectors such as adenovirus, adeno-associated virus (AAV), retrovirus and lentivirus have been widely used for delivering transgenes. The non-integrative nature of adenoviral transgenesis may not be ideal for longterm gene therapy9. Immune response and limited cargo space also precludes the extensive use of adenoviral vectors. On the other hand, integrative lentiviruses and retroviruses ensure the permanency of transgene expression. However, immune response and limited cargo space is still a major drawback10,11. The possibility of the viral elements reconstituting the active, self-replicating viral form is also a concern12,13. Despite these drawbacks viral systems remain the vector of choice if efficient transgenesis is required14. On the other end of the transgene vector spectrum is plasmid-mediated transgenesis that is characterized by low efficiency15. However, nonviral vectors are generally considered to be safer than the viral options. Therefore, to fill the niche of a vector that is characterized by highly efficient transgenesis, the ability to integrate large transgenes, and a superior biosafety profile, transposon-mediated delivery systems have been developed. Transposable elements are genetic elements that can move from one place in the genome to another. These naturally occurring elements have been copied to enable the movement of a transgene flanked by inverted terminal repeat sequences from a vector to the genome. Prominent among these ‘‘cut-and-paste’’ transposition systems are Sleeping Beauty, Tol2 and piggyBac16–19. Among them piggyBac system has generated great interest by virtue of its ability to achieve robust, highly efficient transposition even with large transgenes20–23. The successful utilization of piggyBac system to deliver reprogramming constructs for generating induced pluripotent stem cells (iPSCs) from somatic cells has also contributed to the enthusiasm24,25. Transposition has been achieved by delivering the transposase enzyme as protein, mRNA or expression plasmid26–28. When delivered as plasmids, the transposase and the inverted terminal repeat-flanked transgene are usually loaded onto two separate plasmids vectors including the helper plasmid (transposase-bearing) and the donor plasmid (transgene-bearing). In a two-plasmid system, ensuring the co-delivery of both the elements is a challenge; this problem is exacerbated when delivery is less efficient, such as in vivo applications. Therefore, SCIENTIFIC REPORTS | 4 : 7403 | DOI: 10.1038/srep07403 1 www.nature.com/scientificreports attempts have been made to combine these two elements together in a single plasmid for efficient co-delivery29,30. Excitement of using transposons for gene delivery has been tempered by the apprehension of heightened levels of genotoxicity and mutagenesis due to prolonged expression of transposase. This concern is based on the prospect of the transposon element continually hopping from one place in the genome to another under the influence of the continued presence of transposase31. There is also an apprehension that the lingering transposase would remove the already integrated transgenes, as not all transposons reenter the genome32. Moreover, prolonged expression can provoke an immune response to the foreign transposase that may prevent any subsequent re-administration. Therefore, it is prudent to inactivate the transposase gene once it has completed its function of transgene integration. Most studies using piggyBac have depended on the transposase-bearing plasmid to be lost through dilution following cell division. However, the cell division rates can vary widely in different tissues and hence this process of transposase dilution is not reliable. Delivering transposase in the form of plasmid also risks random integration of the transposase element into the genome33. This can happen from nonspecific, nuclease-mediated linearization of the plasmid, or from the formation of sheared linear forms during the preparation of the plasmid. These linear forms can integrate randomly into the genome, thereby perpetuating transposase expression in some cells even when most of the circular forms get diluted out due to cell division. The co-delivery of both transposase and transposon from a single plasmid has added another level of complexity to the problem of transposase integration; there might be heightened prospect of transposase integration due to the formation of transient linear forms after transposition (Figure 1A). A promising solution to this problem was suggested by Urschitz et al.29, in which the promoter for the transposase is included in the transposon, within the inverted (...truncated)