Introduction

The Sleeping Beauty (SB) transposon system [1] is a non-viral, integrating vector that is an alternative to viral vectors for gene therapy [2–5]. The SB system consists of two parts: the transposon, which is defined by inverted terminal repeats and which carries an expression cassette for the therapeutic gene, and a second expression cassette for the transposase enzyme (Figure 1A). When expressed, the transposase excises the transposon with its cargo from a plasmid and inserts it into the chromosomal DNA of the host. In practice, transposition results in stable integration of the transgene into a vertebrate host genome and prolongs expression of transgenic products such as Factor IX, β-glucuronidase and alpha-L-iduronidase (IDUA) [3,6–8] compared to most [9] non-integrating plasmid vectors.

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Figure 1. Expression of IDUA from different promoters in NOD/SCID MPS I mice.

A. Vectors used in this study. The T2 SB transposon vectors (inverted arrowheads) contained either a “ubiquitous” CAGGS promoter or the liver-specific promoter (LSP) ApoEHCRhAAT. pKT2/ApoEHCRhAAT-hIDUA and pT2/miniCAGGS-hIDUA have different backbones and the selectable markers, kan and amp, respectively. B. Kinetics of hIDUA expression from LSP and CAGGS promoters in NOD.129(B6)-Prkdc^scidIDUA^tm1Clk/J (NOD/SCID) mice. Symbols: +SB11 females, filled red circles and solid lines; –SB females, open red circles and dotted lines; +SB11 males, filled blue squares and solid lines; –SB males, open blue squares and dotted lines. Each point represents the mean hIDUA activity ± SD, n=6. The CAGGS graph in panel B, is from Figure 1d of [8]. C. Comparison of transposition efficiency of SB11 and SB100X transposases in C57BL/6 mice by the excision product (EP) assay. 2μg of either pCMV-SB11 or pCMV-SB100X transposase-encoding plasmid was co-delivered with 15μg of pT2/CAGGS-GUSB [7] transposon plasmid. DNA for excision assay was isolated from livers 5 days p.i.

https://doi.org/10.1371/journal.pone.0078161.g001

We have shown in immunodeficient NOD.129(B6)-Prkdc^scidIDUAtm1Clk/J mice that the SB transposon system is efficacious in gene therapy of murine mucopolysaccharidosis type I (MPS I) [8], an inherited systemic disorder caused by IDUA deficiency [10]. However, the NOD/SCID MPS I mouse model (www.jaxmice.com), has drawbacks for studying both transposition and correction of MPS I because of impaired double-strand break repair that severely reduces transposition activity [11] and a shortened lifespan with mild clinical manifestations. However, evaluation of treatment effects in immunocompetent MPS I mice so far has not been feasible due to highly variable expression of IDUA following delivery of SB plasmids [7].

In our modified approach, we sought to minimize expression in antigen-presenting cells (APC), increase transposition efficiency by using the hyperactive transposase SB100X, and provide a more effective and less toxic cyclophosphamide regimen by transiently immunosuppressing mice at the time of vector administration. In addition, we aimed at reducing promoter shutdown and gender bias in IDUA expression associated with the CAGGS promoter [8]. As a result, we have attained sustained expression of IDUA in the liver at levels greater than 100-fold wildtype (WT) for one year in at least half of treated mice.

SB-mediated transgene expression is affected by many variables. The efficiency of transposition depends on the transposon and the transposase sequences, which are consistently being improved [2,5]. Since its first use as gene delivery vector [1], the SB transposase has been re-engineered from SB10 [12] through SB11 [13] and other intermediates [14] to the extremely hyperactive SB100X [15]. Likewise, the structure of the original transposon, T, has been re-engineered for greater activity, e.g. T2 [16] and other versions [17]. Besides the transposon system itself and its cargo, factors affecting expression are associated with the properties of expressed proteins, target cells or organs, delivery procedure, etc. [18]. Promoters regulating SB transposase and therapeutic genes can be either ubiquitously expressed or tissue-specific, either prone or resistant to silencing, and drive different levels of transgene expression. The expressed protein can either be restricted to the producer cell, e.g., luciferase [19], or it can escape the cell and be available for recapture by other cells as is typically the case with lysosomal hydrolases such as IDUA [10]. Host-related factors such as genetic background, gender and age can affect expression of the transposed gene. For example, we found that IDUA expression from the CAGGS promoter was silenced in female mice compared to male mice [8]. To gain insights into which variables affecting transgene expression lead to its loss and which facilitate stabilization of IDUA activity at therapeutic level, we examined decay rates in SB-mediated IDUA activity profiles obtained under a variety of conditions. We reasoned that these rates are by and large a reflection of various influences on transgene expression. We found that immune responses are the most important variable to control in order to prevent loss of transgene expression [20].

Results

Expression of hIDUA in C57BL/6 mice over one year

Without immunosuppression, IDUA activity was lost in about 90% of IDUA vector-treated mice by 4-8 weeks post-injection (whether or not SB transposase-encoding plasmid was co-infused (±SB, Figures 2A and B). Cyclophosphamide (CP) treatment was used for immunosuppression in a regimen that consisted of four doses (120 mg/kg i.p.), -24, -6, +24 and +48 hrs relative to the hydrodynamic injection. The time-course of IDUA expression within each cohort of mice was not uniform (Figures 2C-F and Table 1). In four out of eight female mice treated with 5μg transposon-plasmid and in four out of six mice treated with the 25μg transposon-plasmid (+SB), IDUA activity was sustained for one year at more than 100-fold WT activity (Figures 2C and E), a level that is therapeutically corrective for MPS I mice [8]. Increasing the transposon dose from 5μg to 25μg DNA resulted in an initial approximately 5-fold increase in IDUA activity; however, the latter declined and stabilized by eight weeks post-infusion at levels that were not significantly higher than those attained with the 5μg dose (averages of 456- and 404-fold WT, respectively). In mice that did not receive the transposase plasmid (-SB), IDUA generally declined gradually over a one-year period, ending with an average activity level significantly below the therapeutic range (Figures 2D and F). Some mice appear to have been insufficiently immunosuppressed based on the rapid decline in IDUA expression after 4-36 weeks (Figures 2C, D and F, asterisks). Examples of gradual decline despite treatment +SB are indicated (Figures 2C and E, 1, 6 and 24, arrows).

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Figure 2. Expression of transgenic hIDUA in C57BL/6 mice.

Mice aged 12 weeks were treated with 5μg or 25μg pKT2/LSP-IDUA ± pCMV-SB100X at 5:1 and 10:1 weight ratio, respectively. Plasma IDUA activity in untreated controls (WT) was 10±4 nmol/ml/hr. The dashed double-lines indicate the therapeutic level [8]. Arrows indicate expression profiles of +SB-treated mice with gradual or incomplete loss of IDUA activity. Asterisks indicate rapid decay in CP-immunosuppressed mice. Numbers indicate mouse IDs as listed in Table 1.

https://doi.org/10.1371/journal.pone.0078161.g002

Mouse ID	Profilea	Plasma IDUA,	Frequency, DNA copy/cellb
		x WTc	IDUA	EP	SB
5μg, +SB (Figure 2C)
1	G	20	0.04	bdld	0.02
2	S	768	0.32	0.13	0.11
3	S	148	0.14	0.03	0.03
4	R	1	bdl	bdl	Bdl
5	R	1	bdl	bdl	Bdl
6	R	1	bdl	bdl	bdl
7	S	107	0.04	0.02	0.02
8	S	592	0.08	0.03	0.03
eMean±SD for S		404±330	0.15±0.12	0.05±0.05	0.05±0.04
5μg, –SB (Figure 2D)
9	R	1	bdl	bdl	bdl
10	G	17	0.58	bdl	bdl
11	G	34	0.45	bdl	bdl
12	R	2	bdl	bdl	bdl
13	G	25	0.09	bdl	bdl
14	G	19	0.37	bdl	bdl
15	G	8	0.15	bdl	bdl
16	G	22	0.52	bdl	bdl
17	R	3	bdl	bdl	bdl
18	G	10	0.21	bdl	bdl
fMean±SD for G		19.3±8.9	0.34±0.19	bdl	bdl
25μg, +SB (Figure 2E)
19	S	178	0.09	0.02	bdl
20	S	801	0.35	0.24	0.03
21	R/S	7	bdl	bdl	bdl
22	S	1051	0.59	0.22	0.03
23	S	164	0.08	0.02	bdl
24	G/S	84	0.07	bdl	bdl
eMean±SD for S		456±440	0.28±0.24	0.13±0.12	0.03
25μg, –SB (Figure 2F)
25	G/R	1	bdl	bdl	bdl
26	G	40	0.52	bdl	bdl
27	G/R	1	bdl	bdl	bdl
28	G	35	0.26	bdl	bdl
fMean±SD for G		37.5±3.5	0.4±0.2	bdl	bdl

Table 1. IDUA expression profiles and activity in plasma vis-à-vis transposition and transgene/plasmid maintenance in liver of immunosuppressed C57BL/6 mice one year post-treatment.

aIDUA expression patterns are as defined in the Results: R-rapid decay; G-gradual decay; S-stable;R/S and G/R are hybrid profiles and G/S is very slow gradual decay; in R/S and G/S stabilization not confirmed;

bqPCR determination of copy numbers of DNA sequences of human IDUA and SB100X (transposase) transgenes or excision/repair product plasmids;

cIDUA activity was assayed in plasma; WT in C57BL/6 mice,10 nmol 4MU/ml/h;

dbdl=below detection limit;

eMean±SD calculated for mice with stabilized expression;

fMean±SD calculated for mice with gradual expression.

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Maintenance of transgenes and transposition in the liver

Whenever IDUA activity was rapidly lost (i.e., half-life of less than 3 days), neither transgenes nor plasmid sequences could be detected in samples of mouse liver by qPCR (Table 1). This suggested that in these cases, cells that initially took up and expressed the transgenic DNA were eliminated from the liver. In contrast, if the expression profile of transgenic IDUA was stable, or declined at a moderate rate, then we could always measure the IDUA transgene level by qPCR. Notably, plasmids that remained after transposition of the transposon cargo, i.e., EP, were detected only in mice with stabilized, high-level IDUA activity (Table 1). This observation supports our hypothesis that sustained, therapeutic-level transgene expression can be achieved after transposition. EP copy numbers per cell were not significantly different in mice dosed with either 5μg or 25μg of transposon (0.05±0.05 and 0.13±0.12, respectively); the variability in copy number may reflect episomal survival in liver cells one year post-injection. There was no evidence of EPs in mice whose IDUA activities gradually declined over a year (Table 1), although IDUA transgenes were detected at essentially the same frequency as in those with stabilized activity. Normalizing IDUA activity to IDUA transgene copy number for the non-saturating expression conditions of 5μg of transposon (404/0.15 = 2700 for +SB and 19/0.34 = 57 for –SB, Table 1) indicates that without SB transposase, expression of the transgenes was about 48-fold lower than with transposase (Figure 3A). Transposition efficiency, reflected by the EP/IDUA ratio for both doses of transposon was 38±2% (n=8) in mice with sustained IDUA expression (Figure 3B), i.e., about one-third of plasmids that survived as episomes in the liver after one year, had donated a transposon for integration. Our data from the 5μg transposon doses suggest that expression from episomes is about 130-fold (48/0.38) weaker than expression from transposed IDUA expression cassettes, i.e., expression of IDUA genes from episomes is about 1% that from chromosome.

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Figure 3. IDUA transgene activity is enhanced when transposition is supported by SB transposase.

A. Plasma IDUA activities were normalized to IDUA copy per cell number. SB-treated mice with stabilized IDUA activity were compared with mice that were infused with only the transposon plasmids and did not lose IDUA activity in a rapid decay. The IDUA activity/copy number ratio, mean±SD, in arbitrary units, was +SB, 27000±19600 (n=8) and –SB, 900±800 -SB (n=9); **p=0.001. B. Transposition efficiency in mice with stabilized expression is calculated as the copy number ratio of pKT2/ApoEHCRhAAT-hIDUA plasmids that underwent transposon excision (EP, copy per cell) to the total number of pKT2/ApoEHCRhAAT-hIDUA (IDUA, copy per cell); mean EP/IDUA±SD =38±16%. Data from mice treated with 5μg and 25μg transposon doses were pooled.

https://doi.org/10.1371/journal.pone.0078161.g003

Kinetics of loss of IDUA expression

Our inspection of transgene expression profiles under many different experimental conditions indicated that there are several components that account for loss of transgene expression. Quantified decay rates of LSP-IDUA expression from individual mice could be divided into three profiles: rapid decay, T_1/2 =1.6±0.5 days (n=17) calculated from all mice treated without immunosuppression (Figure 4, red solid line; data in Figures 2A and B); gradual decay, T_1/2= 91±49 days (n=7), typical in CP-immunosuppressed mice that did not receive the transposase-encoding plasmid (Figure 4, blue line; data in Figure 2D); and sustained expression that was observed only in those immunosuppressed mice that received the SB-transposase-expressing plasmid (Figure 4, black line; data in Figure 2C). In those mice that lost expression at unpredictable times (Figure 4, red dashed lines; mice 25 and 27 indicated by asterisks in Figure 2F), the half-lives of expression were within the rapid decay range observed in animals that were not treated with CP.

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Figure 4. Summary of quantitative evaluation of transgene expression in mice following hydrodynamic delivery to the liver.

Three decay rates can account for all of the profiles of expression that we have observed under about 128 different combinations of experimental variables. These decay rates are: initial (i), associated with promoter silencing [8], green line; rapid (ii), associated with loss of plasmids and transgenes, red line; and gradual (iii), blue line. Stabilized expression (iv), black line, at levels >100WTdeemed therapeutic [8] was attained only with LSP, transposition using SB 100X transposase and a transient 4-dose immunosuppression with CP administered intraperitoneally around the time of hydrodynamic delivery. We hypothesize that the delayed loss of expression (red dashed lines, mice 25 and 27 in Figure 2F), that has the same half-life of <3 days, is rapid as (ii).

https://doi.org/10.1371/journal.pone.0078161.g004

Rapid decay at its earliest onset occurred between one and two weeks post-infusion, which is consistent with the onset of an adaptive immune response in the absence of immunosuppression by CP. Note that our T_1/2 values have an inherent imprecision due to the infrequency of plasma IDUA measurements, which prohibits specific identification of the timing of initiation and termination of the decay process. We evaluated IDUA loss-of-expression rates obtained from our earlier studies using a CAGGS-IDUA expression cassette [7,8]. Compared to the LSP expression profiles, with CAGGS there was a gender-biased initial decay, steep in females and more gradual decay in males, during the first 3 days (Figures 1B and [8]). Thus, despite differences between CAGGS and LSP-expression due to gender bias and promoter silencing associated with CAGGS, the rates of final elimination of IDUA expression (T_1/2=1-2 days) were similar for the LSP and CAGGS promoters, as was clearance of transgenes and excision products in those mice where all activity was lost.

Discussion

Achieving effective, life-long gene therapy is daunting because many variables can affect transgene expression. Such is the case with SB-mediated gene therapy for MPS I, where the goal is sustained, high-level IDUA expression in the liver.

In our previous work with the NOD/SCID MPS I model, IDUA expression profiles within a treatment group were consistent whereas high variability and inconsistency of expression were evident in immunocompetent MPS I mice even when immunosuppressed. This study, which employed delivery of immunogenic human IDUA-encoding transposons to immunocompetent mice, is a good model in which to examine the variable kinetics of transgene expression that one would expect in immunocompetent IDUA-deficient animals. Our achievement of sustained, high-level IDUA expression in 50% of C57BL/6 mice (Figures 2C and E) mimics what we have observed when treating MPS I mice. Our best results were obtained using the complete transposon system +SB100X transposase, a liver-specific promoter, and a single, four-dose regimen of CP immunosuppression around the time of transgene delivery. While the two former modifications to minimize immune responses are obvious, demonstration of SB-mediated effects on expression over one year has not been reported. That higher levels of SB activity were indeed attained is shown by quantitative measurements of excision circles (Figure 1C), and the levels of plasma IDUA following SB transposition (e.g., Figure 2C) are higher than those we have previously reported [7,8]. Our data suggest that transposition of the transgenic expression cassette into chromatin is necessary for long-term activity following plasmid delivery to the liver. However, stable transgene expression has been reported for minicircles [9,25–27] and mini-intronic plasmids [28], presumably because they either lack sequences that lead to transcriptional silencing [9,27] or are contained in an intron within the eukaryotic expression cassette [28]. Notably, in effectively immunosuppressed C57BL/6 mice, LSP-driven expression from episomes might continue for months, although we have never observed stabilized expression from episomes at >100 WT and the resulting episomal IDUA expression levels after one year never reached those deemed therapeutic for MPS I [8]. These observations are consistent with our calculations that expression from whole plasmids is about 1% that from transposed expression cassettes. They are also similar to what we saw upon Cre recombinase-induced inactivation of non-transposed erythropoietin transposons [29].

Prolonged persistence of low-level transgene expression from episomes may explain the absence of a clear-cut distinction in transgene activity between chromosomally integrated compared to episomal transposons in shorter-term experiments. By extending our studies out to one year, the long-term effects of using the complete SB system to achieve chromosomal integration of the IDUA expression cassette became obvious. Transposon doses of 5μg and 25μg yielded comparably high levels of stabilized transgenic IDUA activities (Figure 2C and E, Table 1). Although the increase of transposon+transposase dose from 5+1μg to 25+2.5μg, respectively, resulted in the expected approximately 5-fold increase in day-1 IDUA activity, there was a decline in average transgene expression in the 25+2.5μg dosed mice that ranged from 5 to 20-fold from peak activity at stabilization around 8 weeks p.i. (ibid). We interpret the gradual initial declines in +SB and –SB mice dosed with 25μg transposon-plasmid as being due to innate immune responses to a higher concentration of plasmid sequences, which can trigger transcriptional repression [9,25,27]. Thus, “more may be less” in the case of loading hepatocytes with transgenic constructs that remain as episomes.

Comparison of expression levels indicates the importance of carefully choosing the transgene promoter. IDUA activity from LSP was at therapeutic levels without the drawback of the initial fast shutdown of expression from CAGGS in females, which occured in both C57BL/6 and NOD/SCID mice [8] (Figures 1B and 4). However, the liver-specific promoter alone is not a solution against loss of expression, even with transposition. Although liver-specific (i.e., hepatocyte-specific) promoters minimize expression from APCs, overexpressed IDUA may not evade a robust immune system. Thus, while LSP-IDUA expression is not prone to silencing, LSP does not rescue expression from elimination by adaptive immune responses. Without immunosuppression, rapid decay, (Figures 2A and B) occurred at a time consistent with the onset of the adaptive immune response, 7 to 14 days post-infusion. Osborn et al. [28] demonstrated the presence of anti-IDUA IgG antibodies in the sera of mice treated with minicircle-encoded human IDUA without costimulatory blockade. However, it appears that cell-mediated responses are responsible for ultimately eliminating IDUA expression in both their experiments and in ours. These observations parallel ours in non-immunosuppressed mice that received CAGGS-IDUA; CTL-mediated immune responses removed transduced hepatocytes throughout the liver [7]. In this study, such rapid loss was evaded in over 70% of the mice treated with LSP-IDUA and transient CP regimen.

There are two plausible explanations for complete rapid loss of transgene expression in mice that were immunosuppressed: 1) some mice are resistant to immunosuppression by CP and 2) some intraperitoneal injections failed due to inadvertent placement of the injection other than intraperitoneal cavity [30,31]. Partial failure of immunosuppression with a 4-dose regimen could explain some of the outlier profiles of IDUA expression that we observed (Figures 2C and E, arrows). If loss of activity in the first six weeks in the 5μg and 25μg transposon-dosed groups was due to failed immunosuppression, then nearly 75% (8/11) of the truly immunosuppressed mice had stabilized expression of IDUA at therapeutic levels. If this is the case, our data suggest that improvement of immunosuppression will lead to sustained therapeutic effects in most mice.

In conclusion, in this and our previous studies [7,8,32] we have examined the levels of transgene expression for up to one year in transposon-injected mice under about 128 different combinations of SB transposases, transgene promoters, mouse genetic backgrounds, mouse gender, and cyclophosphamide immunosuppression. Broadly, two basic phenomena lead to the loss of transgene expression – presumably, transcriptional silencing and adaptive immune responses. Our quantitative analyses allowed us to discern the different kinetics underlying loss of transgene expression and better interpret experimental results. These causes can be divided into the ones that are feasible to control (e.g., choice of promoter, immunosuppression protocol, plasmid dose) and those that are poorly understood. We conclude that despite what looks like a large variation in patterns of transgene expression, many combinations of factors affecting transgene expression lead to only a limited number of post-treatment scenarios (Figure 4). The SB system is capable of providing effective gene therapy in mice, but improved methods of immunosuppression are needed to increase the percentage of treated animals that do not lose transgene expression.

Materials and Methods

Acknowledgments

We thank Mark Kay for the gift of pAAV-hFIX 16 plasmid containinΥthe liver-specific promoter, and Vincent Keng for the gift of primers for SB11/100X transposases; Erik Olson for technical assistance; Michael Klug, Kelly Podetz-Pedersen, Jeff Reineke and Dan Wolf for helpful discussions.