Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

Abstract

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals.

Figure 1

The Sleeping Beauty (SB) transposon and its transposition. (A) Structure of the terminal repeats of the SB transposon. The DRs of the ITRs are designated by arrowheads and are labeled according to their positions in the transposons used in this study. The boxed TAs flanking the transposon result from duplication of the original TA insertion site. (B) “Cut-and-paste” mechanism of SB transposition revised from Luo et al. [14] and Plasterk et al. [2]. The two major steps involved in transposition, excision and integration of a transposon are shown. The two broken ends at the donor sites, joined together by non-homologous end-joining (NHEJ) enzymes encoded by the host, leave a footprint at the donor site

Figure 2

Schematic of the excision assay. Plasmids containing a transposon and CMVSB transposase were co-transfected into HeLa cells. Four days post-transfection, cell lysates were obtained and used for PCR with primers flanking the donor sites. The PCR products were sequenced to determine the footprints of the excision. The procedure is shown on the left and the state of the transposon and its excision product is shown on the right

Figure 3

PCR analysis of transposon excision from plasmids in HeLa cells (A and B) and in zebrafish embryos (C). (A) Plasmids with transposons and CMVSB transposase (pT/neo and pCMVSB) were co-transfected into the HeLa cells. Cell lysate was obtained for nested-PCR using primers outside the transposon. ΔDDE is a transposase without a catalytic domain. (B) Time-course accumulation of excision products from HeLa cells. Hours post-transfection are marked on top of the gel. Each time point is represented by two separate transfections. The marker lane (M) on the left of the gels in A and C is a 100-bp ladder (New England Biolabs). (C) SB mRNA was co-injected into one-cell-stage zebrafish embryos with plasmids containing a transposon (pT/neo). Twenty-four hours after microinjection, lysates from single embryos were used for PCR analysis. Two different embryos were used in the last three categories. *: pT/neo plasmids mixed with embryo lysate were used as template in this case

Figure 4

Quantification of relative excision activity in HeLa cell excision assay. Excision levels of four IR/DR mutations were measured relative to the activity of LoLi–RiRo (the pT/HindIIIneo transposon). The top gel shows excision PCR products run on a 6% polyacrylamide gel stained with SYBR green I. The lower gel shows PCR amplification of a segment of the backbone of pT/neo and pCMVSB (or pSB10-ΔDDE) as an input control for the total plasmid in the lysate. The relative excision abundance was measured as a ratio of the band intensity of the excision PCR products to that of the amplification of the segment on the plasmid backbone. “Rel. template” indicates the relative amount of the input lysate. Relative excision activity (“Rel. activity”) is indicated as a percentage of control activity using the ratio for each mutation compared to a standard curve derived from the ratio of the different dilutions of the original pT/HindIIIneo activity. ND, non-detectable

Figure 5

Terminal nucleotides in the outer DRs are important for excision. (A) Sequence comparison of Lo and Li. Mutated sequences in regions I and II are underlined. (B) Mutations in region I at the first and third positions at the terminus of Lo are underlined. Lo(CAG)Li–Ri(CTG)Ro indicate the pT/HindIIIneo transposon. Quantification of the excision activity is as described in Figure 4. ND, non-detectable. (C) Mutations in region II wherein three out of the five terminal base pairs (TTAAG to GGGAG) were changed

Figure 6

Effect of TA-dinucleotides on excision. (A) Excision analysis of TA mutations on either side and both sides of the transposon. TA Lo-RoTA indicates the pT/HindIIIneo transposon. Mutations are underlined. (B) Sequences of the excision site of the mutated transposons. The footprints are underlined. ND, non-detectable

Figure 7

Excision products in the livers of SB-transposon-treated MPS VII mice. Lane 1, 100-bp interval markers; lane 2, treatment with pT/CAGGS-GUSB alone; lane 3, treatment with pT/CAGGS-GUSB + pCMVSB, lane 4, sham-treatment with pBluscript