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Minimal Piggybac Vectors for Chromatin Integration

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Minimal Piggybac Vectors for Chromatin Integration Gene Therapy (2014) 21,1–9 & 2014 Macmillan Publishers Limited All rights reserved 0969-7128/14 www.nature.com/gt ENABLING TECHNOLOGIES Minimal piggyBac vectors for chromatin integration V Solodushko1,2, V Bitko3 and B Fouty1,2,4 We describe novel transposon piggyBac vectors engineered to deliver transgenes as efficiently as currently available piggyBac systems, but with significantly less helper DNA co-delivered into the host genome. To generate these plasmids, we identified a previously unreported aspect of transposon biology, that the full-length terminal domains required for successful plasmid- to-chromatin transgene delivery can be removed from the transgene delivery cassette to other parts of the plasmid without significantly impairing transposition efficiency. This is achieved by including in the same plasmid, an additional helper piggyBac sequence that contains both long terminal domains, but is modified to prevent its transposition into the host genome. This design decreases the size of the required terminal domains within the delivered gene cassette of the piggyBac vector from about 1500 to just 98 base pairs. By removing these sequences from the delivered gene cassette, they are no longer incorporated into the host genome which may reduce the risk of target cell transformation. Gene Therapy (2014) 21, 1–9; doi:10.1038/gt.2013.52; published online 17 October 2013 Keywords: piggyBac; mutagenesis; stable gene delivery INTRODUCTION integrated into the host cell genome, they perform no useful Transposon vectors are a proven and viable alternative to viral function. They may potentially increase the risk of cell vectors for stable gene delivery.1–4 Like integrated viruses, transformation due to their retained promoter and enhancer transposons deliver transgenes to target cells in vitro and in vivo activity, however. Attempts to reduce the size of the terminal where they are incorporated into the host genome. Unlike viruses domains to decrease this potential risk of transformation have 9,13 they do not generate an immune response, they have a simpler resulted in a significant loss of transposition efficiency. genome, and are easier to handle. In addition, they can hold a In this paper, we describe a piggyBac vector in which most of significantly larger transgene insert than viruses, in some cases up both terminal domains have been removed from the delivery to 100 kilobases.5 These characteristics make transposons an cassette without a significant loss of transposition efficiency. Only attractive option for gene delivery. two minTR sequences remain within the delivery cassette, both of PiggyBac vectors are one of the most active and flexible which are very short (35 base pairs for 50 minTR and 63 base pairs transposon systems available for the stable transfection of for 30 minTR). The expected negative effect on transposition of mammalian cells.6,7 The wild-type piggyBac transposon is 2472 deleting most of the terminal domains is compensated by the base pairs in length, and is composed of two inverted minimal presence of a transposition-incompetent piggyBac transposon terminal repeats (minTRs), two internal domain sequences and a containing both long terminal domains and the transposase gene transposase-encoding domain8,9 (Figure 1, wild type). Transposase within the helper part of the plasmid. This design contains two catalyzes the excision of the transposon from one DNA different sets of piggyBac sequences each of which has been source (that is, a delivered plasmid) and allows its subsequent modified to serve different functions. The working (transposable) re-integration into another DNA source (that is, the host cell transposon has been truncated to decrease the amount of extra genome). In the majority of piggyBac vectors, the transposase DNA incorporated into the host genome, whereas the helper (non- gene has been removed from the transposon, replaced by transposable) sequence provides the long terminal domains transgenes of interest; the transposase is then usually delivered necessary for efficient transposition of the working transposon. to the cell by a separate plasmid, a technique first demonstrated This design decreases the size of the required terminal domains in insect cells10–12 (Figure 1, delivery and helper plasmids). within the delivered gene cassette from about 1500 to just 98 base The minTRs and internal domains are crucial for the effective pairs which significantly decreases the size of the integrated integration of the transposon into the host genome and together sequence within the host cell genome. (known as terminal domains) consist of more than 700 base pairs each.9,13–16 The 50 terminal domain also serves as a native promoter for transposase expression. During transposition, the RESULTS terminal domains are integrated into the host cell genome, Full-length terminal domain sequences do not need to be in the exclusively at TTAA integration site, alongside the delivered delivered transposon for efficient transposition of piggyBac vectors transgene of interest as part of the transposon.11,14 Therefore, like We hypothesized that most of the terminal domain sequences integrated viruses, they deliver a significant amount of extra DNA could be moved outside of the transposon with minimal loss of to the target cell genome. These sequences (the terminal transposition efficiency. To test this hypothesis, we designed domains) are required for successful transposition, but once several plasmids (Figure 1) and determined their transposition 1Center for Lung Biology, University of South Alabama School of Medicine, Mobile, AL, USA; 2Department of Pharmacology, University of South Alabama School of Medicine, Mobile, AL, USA; 3NanoBio Corporation, Ann Arbor, MI, USA and 4Department of Internal Medicine University of South Alabama School of Medicine, Mobile, AL, USA. Correspondence: Dr V Solodushko, Center for Lung Biology, Department of Pharmacology, MSB 3406, University of South Alabama School of Medicine, Mobile, AL 36688, USA. E-mail: [email protected] Received 1 May 2013; revised 16 July 2013; accepted 27 August 2013; published online 17 October 2013 Minimal piggyBac vectors V Solodushko et al 2 Figure 1. Schematic presentation of vectors. 50 TRmin, 30 TRmin: minimal 50 or 30 terminal repeats (in black); ½ 30 minTR: half of the 30 terminal repeat in plasmid-166 (in black); 50 TD, 30 TD: 50 or 30 full-length terminal domains (including the 50 TRmin or 30 TRmin) (in yellow and black); transgene: delivered gene(s) (in this paper – RFP, (in blue)); transposase: piggyBac transposase gene (in red). efficiency in target cells. The first plasmid contained a delivered promoter (plasmid-200) or a plasmid carrying the entire wild-type cassette encoding the reporter gene, red fluorescent protein (RFP), piggyBac (p3E1.2) failed to substantially increase transposition flanked by 50 and 30 minTRs (plamid-132). In a second plasmid, we efficiency (0.13 and 0.11%, respectively). Transfection with inserted a wild-type piggyBac transposon separated from the RFP plasmid-137, however, resulted in a marked increase in the delivery cassette by 683 and 2466 base-pair linkers (plasmid-137, number of cells stably expressing RFP at 28 days to 3.89%. These Figures 1 and 2b). This construct allowed us to add full-length results suggested that minTR alone are not sufficient to allow terminal domains back into the plasmid without including them plasmid-to-chromatin transposition, but the presence of full- within the RFP-delivered cassette. The presence of piggyBac length terminal domains elsewhere in the plasmid, even if they transposase in the wild-type transposon (driven by its native are located outside of the delivery cassette, will allow successful promoter) eliminated the necessity of using a helper vector to transposition into the host cell genome. Delivery of full-length deliver the transposase. A third plasmid (plasmid-185), also terminal domains in a separate plasmid, as was shown with contained the RFP-delivery cassette, but included an additional p3E1.2, failed to support the transposition. (modified) full-length transposon in which both TTAA integration These results with plasmid-137 did not clarify whether only the sites were mutated (to GTAA) to prevent transposition of the RFP-delivery cassette was integrated into the host cell genome or full-length piggyBac into the host genome. In plasmid-185, the whether the entire fragment, containing both piggyBac transposons, full-length terminal domains of the second transposon were was delivered. Therefore, we tested the integration efficiency of separated from the minTRs of the RFP delivery cassette by two plasmid-185, a plasmid in which the TTAA integration sites flanking linkers of 254 and 3354 base pairs. As the activity of the native the full-length piggyBac vector in the helper part of the plasmid transposase promoter is unpredictable in many mammalian were mutated (to GTAA), preventing its excision from the plasmid cells,17 we replaced the native promoter with an SV40 promoter and thus preventing its integration into the host (other modifica- in this, and subsequent, plasmids (promoters are not shown in tions are described in Figure 2b). Plasmid-185 had significantly Figure 1, refer to Figures 2–4 for plasmid details) to more reliably greater transposition efficiency than plasmid-137 (13.4 versus 3.89% drive expression of the transposase. This replacement necessitated of initially transfected cells at 28 days) (Figure 2a). The transposition a partial duplication of the 50 terminal domain to
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