Development of Hyperactive Sleeping Beauty Transposon

Development of Hyperactive Sleeping BeautyTransposon Vectors by Mutational Analysis

Hatem Zayed,1 Zsuzsanna Izsvak,1,2 Oliver Walisko,1 and Zoltan Ivics1,*1 Max Delbruck Center for Molecular Medicine, Robert Rossle Strasse 10, D-13092 Berlin, Germany

2 Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary

*To whom correspondence and reprint requests should be addressed.

Fax: +(49) 30 9406-2547. E-mail: [email protected].

The Sleeping Beauty (SB) transposable element is a promising vector for transgenesis invertebrates and is being developed as a novel, nonviral system for gene therapeutic purposes.A mutagenesis approach was undertaken to improve various aspects of the transposon, includingsafety and overall efficiency of gene transfer in human cells. Deletional analysis of transposonsequences within first-generation SB vectors showed that the inverted repeats of the element arenecessary and sufficient to mediate high-efficiency transposition. We constructed a ‘‘sandwich’’transposon, in which the DNA to be mobilized is flanked by two complete SB elements arrangedin an inverted orientation. The sandwich element has superior ability to transpose >10-kbtransgenes, thereby extending the cloning capacity of SB-based vectors. We derived hyperactiveversions of the SB transposase by single-amino-acid substitutions. These mutations actsynergistically and result in an almost fourfold enhancement of activity compared to the wild-type transposase. When combined with hyperactive transposons and transiently overexpressedHMGB1, a cellular cofactor of SB transposition, hyperactive transposases elevate transposition byalmost an order of magnitude compared to the first-generation transposon system. Theimproved vector system should prove useful for efficient gene transfer in vertebrates.

INTRODUCTION

Considerable effort has been devoted to the developmentof gene delivery strategies for the treatment of inheritedand acquired disorders in humans [1]. These methods canbe broadly classified as viral and nonviral technologies,and all have advantages and limitations. Viral vectors,where available, are efficient at introducing and express-ing genes in cells. However, adapting viruses for genetransfer restricts genetic design to the constraints of thevirus in terms of size, structure, and regulation of expres-sion. Nonviral methods, including DNA-condensingagents, liposomes, microinjection, and ‘‘gene guns,’’might be easier and safer to use than viruses, but arenot equipped to promote integration into chromosomes.As a result, stable gene transfer frequencies using nonviralsystems have been very low.

A relatively new addition to the gene therapist’s tool-box is transposable element-based gene vectors [2,3].Transposons are genetic elements that have the distinc-tive ability to move in genomes. Especially useful forgenetic analyses are members of a class of transposableelements that move via a ‘‘cut-and-paste’’ mechanism:the transposase catalyzes excision of the transposon from

its original location and promotes its reintegration else-where in the genome [4]. The simplest DNA transposonsare framed by terminal inverted repeats (IR) and containa single gene encoding a transposase. The transposase cantrans-mobilize elements as long as they retain the IRs,which forms the basis of powerful experimental manip-ulation. The P transposable element has revolutionizedDrosophila genetics and is widely used as a vector forgerm-line transgenesis and insertional mutagenesis inflies [5]. Until very recently, transposon vectors werenot available for genetic analyses in vertebrates. This isbecause the vast majority of elements currently residingin vertebrate genomes are transpositionally inactive [6–9]. To address this problem, two Tc1/mariner-like ele-ments called Sleeping Beauty (SB) [2] and Frog Prince [10]were reactivated from ancient transposon fossils recov-ered from fish and frog genomes. SB shows efficienttransposition in a variety of vertebrate (including hu-man) cell lines in tissue culture [3,11] and in the mouse invivo, both in somatic tissues [12] and in the germ line[13–17].

Recent experiments from several laboratories havedemonstrated some advantages of SB over the currently

MOLECULAR THERAPY Vol. 9, No. 2, February 2004292Copyright D The American Society of Gene Therapy

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ARTICLE doi:10.1016/j.ymthe.2003.11.024

used viral and nonviral vectors, including stable, single-copy integration [12,18], use of simple plasmid vectors[3,18], and long-term expression of integrated trans-genes at therapeutic levels [12,19–22]. However, in vivotransformation rates with naked SB plasmids adminis-tered through the tail vein into mice were only around5% in the liver [12]. Two immediately obvious areas inwhich the efficiency of SB-mediated gene transfer couldpotentially be improved are the efficiency of vectordelivery and the intrinsic transpositional activity ofthe element itself.

In this work we aimed at a refinement of the SBsystem for higher transpositional activity. Previousimprovements have been made by manipulating eitherthe transposon IRs or the transposase protein. For ex-ample, hyperactive SB vectors have been generated byreducing the length of vector DNA outside the transpo-son in donor plasmids [3] or by the introduction of site-specific mutations into the IRs [23]. However, even theimproved vectors are subject to size restrictions: trans-position frequency of SB decreases with increasinglength of the transposon [3,18,24]. Large (>10 kb) piecesof genomic DNA flanked by two identical copies of Pariselements have been mobilized in Drosophila virilis [25].Paris is a Tc1/mariner-type transposon, which suggeststhat mimicking such a naturally occurring arrangementcould possibly extend the capacity of SB vectors totranspose large DNAs.

Mutations in the SB transposase have been shown toresult in hyperactivity, yielding transposase versionswith an approximately threefold higher activity thanthe wild-type SB transposase [18]. There are severalmechanisms of hyperactivity in transposases. For exam-ple, hyperactive phenotypes of the bacterial elementTn5 are due to the reduction of the self-inhibitoryactivity of intact Tn5 transposase [26], a reduced affinityof an inhibitor protein to the transposase [27], or anincrease in the binding affinity of the transposase to itsbinding sites within the transposon IRs [28]. The com-bination of these three hyperactive mutants yields asynergistic effect, leading to an extraordinarily activetransposase [29]. Interestingly, amino acid replacementsthat change glutamic acid (E) residues to lysine (K) ledto hyperactive transposase versions in three differenttransposon systems, Tn5 [26,28], Tn10 [30], and Himar1[31]. Introducing a proline residue, a secondary structurebreaker, at a defined site in the Tn5 transposase alsoresulted in a hyperactive mutant [27].

In this work we set out to improve SB’s transposi-tional efficiency by modifying the structure of thetransposon IRs and by rationally designed site-directedmutagenesis of the transposase. We constructed a ‘‘sand-wich’’ transposon, in which two complete SB elementsare arranged in a head-to-head orientation. The sand-wich element has superior ability to transpose largetransgenes in tissue culture transposition assays in hu-

man cells. We identified three single-amino-acid substi-tutions that result in hyperactivity of the SB transposase.When combined, these mutations have a synergisticeffect and result in an almost fourfold enhancement ofactivity compared to the wild-type transposase. Togetherwith improved transposon vectors and transiently over-expressed HMGB1, hyperactive transposases boost trans-positional activity to about eightfold compared to thewild-type components of the transposon system. Themodified vector system represents a significant advancein vector development for safe and efficient gene deliv-ery in vertebrates.

RESULTS

Defining the Minimal cis Requirements for SleepingBeauty TranspositionSB has a pair of transposase-binding sites at the ends of itsIRs, a structure termed IR/DR [32] (Fig. 1A). In nature, thetwo functional components of the SB transposable ele-ment are physically linked: the transposase gene is locat-ed between the two IR/DRs (Fig. 1A). However, the twocomponents are separable; a gene of interest can becloned between the IR/DRs, and the transposase can besupplied in trans, expressed from the transposase genelocated either on the same [33] or on a second plasmid [2](Fig. 1A). First-generation SB transposon vectors containDNA sequences of about 120 bp between the left IR/DRand the transposase gene as well as a significant portion(f200 bp) of the transposase coding region [2] (Fig. 1A).This presents a possible safety issue for the application ofthe SB transposon system for gene therapy, because thesesequences can potentially provide sufficient homologyfor recombination with the transposase gene in cells,thereby creating an autonomous, and therefore uncon-trollable, transposable element.

In an attempt to define the minimal cis requirementsfor SB transposition, we subjected the pT/neo transpo-son donor construct (SB transposon marked with a neotransgene) [2] to an analysis in which parts of thetransposable element between the IR/DR and the neotransgene were deleted (Fig. 1B). We cotransfected thedonor constructs with a plasmid expressing the trans-posase (pCMV-SB) into HeLa cells. We placed the cellsunder antibiotic (G418) selection and compared thenumbers of antibiotic-resistant colonies as a measureof transpositional efficiency. Construction of the dele-tion constructs required the introduction of an SpeIrestriction site, which did not lead to major changesin transposition efficiency compared to the pT/neocontrol (Experiments 1 and 2 in Fig. 1B). Further elim-ination of sequences of up to 112 bp still allowedefficient transposition (Experiments 3 to 5), whereascomplete elimination of intervening sequences betweenthe internal transposase binding site and the transgenedrastically reduced transposition efficiency (Experiment

MOLECULAR THERAPY Vol. 9, No. 2, February 2004 293Copyright D The American Society of Gene Therapy

doi:10.1016/j.ymthe.2003.11.024 ARTICLE

6). These results suggest that most of the transposonsequences between the IR/DR and the transgene are notrequired for efficient transposition, but a minimum of8 bp downstream of the internal transposase bindingsite must be retained. We wondered whether this re-quirement is sequence-specific; thus, we replaced the8 bp in the transposon with an unrelated sequence.Incorporation of unrelated sequences nearly restoredwild-type levels of transposition (Experiment 7), suggest-ing a requirement for a spacer region between the trans-posase binding site and the promoter driving theexpression of the neo gene. These results collectivelyestablish that the IR/DRs are sufficient to mediate highlevel transposition.

A Sandwich Arrangement of Two Complete SleepingBeauty Transposons Can Mobilize Large TransgenesSeveral improved versions of the SB transposon have beenengineered [3,23], but all suffer from limitations in insertsize. The Tc1/mariner element Paris has been shown toform mobile, composite elements by flanking long DNAsin an inverted orientation [25]. We reasoned that bymimicking the structure of such composite elements,

the capacity of SB transposase to mobilize long trans-genes might be enhanced.

The sandwich element is a new transposable entity inwhich DNA flanked by two copies of SB is mobilized. Arequirement for such a transposon to work is the inabilityof the individual SB units to transpose on their own. Theterminal nucleotides of the Tc1 element in Caenorhabditiselegans have been previously shown to be required forelement excision [34]. Therefore, we mutated the termi-nal 5V-CA bases of the right IR of pT/neo to 5V-GC (Fig.2A). To test the effects of these mutations on transposi-tion, we compared the activity of the pT*/neo (withmutant right IR) transposon donor construct to that ofpT/neo (wild-type control), using the transposition assaydescribed above. In the negative control, a plasmidexpressing h-galactosidase (pCMVh) replaced the trans-posase. The results showed that the CA ! GC mutationscompletely abolish transposition of T*/neo (Fig. 2B),indicating that transposition of individual SB elementsfrom sandwich constructs can be efficiently inhibited.

All four binding sites within the IR/DR structure (Fig.2A) are required for SB transposition [3]. Therefore, afurther (suspected) requirement for the sandwich trans-

FIG. 1. Definition of the minimal cis requirements for Sleeping Beauty transposition by deletional analysis. (A) Schematic representation of the structural

organization of the two functional components of SB elements. In nature, the gene encoding the trans-acting transposase is physically linked to the cis-acting

terminal inverted repeats (black arrows at the ends of the transposon) that contain the binding sites for the transposase (small white arrows). In the laboratory,

these two components are separated. A gene of interest (such as the neo antibiotic resistance gene) is cloned between the IR/DR repeats, and the transposase is

expressed from the transposase gene maintained on a separate plasmid. Black arrowheads indicate enhancer/promoter elements driving the expression of the

transgenes. In addition to the IR/DR repeats, some sequences upstream of the transposase gene as well as transposase coding sequences are retained in first-

generation SB vectors. (B) Deletional analysis of DNA sequences between the left IR/DR and the SV40 promoter-driven neo transgene. On the left, schematic

drawings show the IR/DR repeats with the transposase binding sites, the neo transgene driven by the SV40 enhancer/promoter, and sequences between the IR/

DR and the transgene that were subject to deletion. The dotted lines represent the deleted sequences in the different constructs. On the right, the transpositional

activities of the different deletion derivatives are shown relative to that of the wild-type transposon vector pT/neo, which was set to 100%.



poson to work is that the transposase should be able tobind to all of its binding sites within the compositeelement. We radiolabeled both the wild-type and themutant IRs and examined their ability to be bound bythe transposase in a mobility shift experiment, usingN123 (the DNA-binding domain of SB transposase) (Fig.2C). The results showed no difference between the wild-

type and the mutant IR fragments in terms of binding toN123 (Fig. 2C, lanes 2 and 4). These results thereforedemonstrate that the induced mutations interfere onlywith the catalytic steps of transposition and not with SBtransposase binding.

Next, marker transgenes were cloned between two T*transposons (containing no transgene) in an invertedorientation, thereby forming a sandwich-like arrange-ment, from which the two individual SB elements cannotexcise but together define a new composite element (Fig.3A). The structure of the sandwich transposon is there-fore as follows: (intact left IR)–body of SB element–(disabled right IR)–insert with a selection marker–(dis-abled right IR)–body of SB element– (intact left IR).Earlier results showed that, although SB was able totranspose transgenes of up tof10 kb, the efficiency oftransposition significantly dropped as the elements gotlonger than 4 kb in length [3]. Therefore, we subcloned a4.7-kb piece of DNA into the sandwich vector to yield atotal transposon length of about 7.7 kb (construct pT/SA7.7 in Fig. 3B). We subcloned an additional 4.5-kbpiece of DNA containing the lacZ gene into pT/SA7.7 toyield a total transposon length of 12.2 kb (construct pT/SA12.2 in Fig. 3B).

We tested the efficiency of transposition of the sand-wich constructs using the in vivo transposition assay andwild-type transposon constructs as controls for compari-son. The sandwich transposon T/SA7.7 jumped about 3-fold more efficiently than the similar-size, wild-typemarker transposon T7.5 and about 2.2-fold more effi-ciently than T6.2, a wild-type transposon that containsthe same transgene insert as T/SA7.7 (Fig. 3B). This resultindicates that the sandwich vector is indeed more effi-cient in transposing relatively long DNA fragments thanwild-type SB. Transposition of the sandwich element T/SA12.2 was still more efficient than that of a 10.3-kb-longwild-type transposon (Fig. 3B). However, the sandwichtransposon apparently abides by the same rule as wild-type SB, namely, that transposition rates are inverselyproportional to the length of the transposon [3] (Fig. 3B).Our results suggest that increasing the numbers of bind-ing sites for the transposase can improve transposition oflarge-size transposable elements and establish the sand-wich element as a useful transposon vector for stableintegration of large transgenes.

Mutagenesis of the Sleeping Beauty Transposase at aLinker Region between the DNA-Binding and theCatalytic DomainsTransposons and their hosts have coevolved and devel-oped strategies that reduce the negative effects on thehost but ensure proliferation of the element [9]. Thus,those elements that were apparently very successful inpropagating themselves within a genome and in colo-nizing new genomes through horizontal transmission,such as Sleeping Beauty [6,7], are unlikely to represent

FIG. 2. Mutations in the right inverted repeat of Sleeping Beauty interfere with

transposition, but not with the binding capacity of the transposase. (A)

Schematic representation of the SB transposon. The transposase gene is

flanked by IR/DR-type inverted repeats (black arrows), which contain the

binding sites for the transposase (white arrows at the ends of the IRs). Two

base-pair changes were introduced at the terminus of the right IR. The C in the

sequence 5V-CAGTTGAAG. . . is the first base of the transposon. (B) A

transposon with mutant right IR cannot transpose. Efficiency of transposition

is assessed as an increase in G418-resistant colony numbers in the presence

(SB) versus in the absence (hgal) of transposase. Numbers are per 3 � 104

transfected HeLa cells. The graph shows that a transposon that has the mutant

IR (pT*/neo) cannot be mobilized by the transposase. (C) Transposase can

bind the mutant IR. Electrophoretic mobility shift assay using 32P-radiolabeled

wild-type (wt) or mutated (IR*) IR fragments as probes and N-123, a derivative

of SB transposase containing the specific DNA-binding domain of the SB

transposase within the N-terminal 123 amino acids.



their most active forms. This predicts that hyperactiveversions of transposases can be generated by mutationalanalysis, which is the case for several transposases,including Tn5 [26–28], Tn10 [30], Himar1 [31], and SB[18]. Mutations into genes can be introduced in either arandom or a site-directed fashion. We explored threedifferent approaches to site-directed mutagenesis of theSB transposase: (1) mutagenesis of a linker region be-tween the DNA-binding and the catalytic domains, (2)replacement of acidic amino acids with basic aminoacids, and (3) incorporation of naturally occurring se-quence variants into the transposase.

The basis of the first approach is that, in the Tn5transposase, there is interference between the C-terminalregion and the N-terminus during interaction with the

transposon DNA [35]. Introduction of a proline residuerelieves this interference by facilitating a conformationalchange, thereby leading to a hyperactive transposase[27,35]. SB transposase consists of an N-terminal DNA-binding domain that mediates interaction with the trans-poson IRs and a catalytic domain responsible for theDNA-cleavage and -joining reactions [2,6,8]. A regionimmediately following the DNA-binding domain has anegative impact on transposase binding to the transpo-son inverted repeats (Fig. 4A), supporting the hypothesisthat it promotes an unfavorable conformation of thetransposase. This region is predicted to assume a helicalconformation and is conserved in the Tc1 family (Fig.4B). Based on the Tn5 observations, we reasoned thatintroduction of proline residues in the predicted helix

FIG. 3. The sandwich Sleeping Beauty vector shows enhanced capacity to transpose long transgenes. (A) Outline of wild-type and sandwich SB transposons. In the

sandwich vector, two complete SB elements flank a transgene to be mobilized in an inverted orientation. The individual SB units cannot transpose due to the

mutations in their right IRs (asterisks). Only the full, composite element can transpose. The small, white arrows are the binding sites for the transposase within the

left (large black arrows) and right (large white arrows) IR/DR repeats. (B) Comparison of the respective transpositional efficiencies of wild-type and sandwich

transposon vectors. Transfections were done in human HeLa cells, and efficiency of transposition is expressed as a ratio of colony numbers in the presence versus

in the absence of transposase. The graph shows that the sandwich transposon vector (black columns) has superior ability over wild-type transposons to integrate

transgenes longer than 7 kb.



motif between the DNA-binding and the catalyticdomains could lead to a change in transposase structure,thereby allowing better access of the transposase to itsbinding sites. Toward that end, we introduced the muta-tions L132P, F134P, T136P, and D140P (only one at atime) into the transposase (Fig. 4B).

Next, we tested the transpositional activities of themutant transposases relative to the wild-type (SB10) trans-posase in the transposition assay. The first three muta-tions essentially abolished transposition, whereas D140Pshowed about 17% of the wild-type activity (Fig. 5A).These results indicate that these amino acid replacementsare detrimental to the activity of the SB transposase,possibly because changing the spatial arrangement of

the transposase domains leads to a nonfunctional struc-ture. Nevertheless, the introduction of less drastic changespossibly by random mutagenesis of this region of thetransposase is a promising strategy for future work.

Amino Acid Replacements That Result inHypertransposing Mutants of the Sleeping BeautyTransposaseBased on findings that some of the Tn5, Tn10, andHimar1 hyperactive mutations are acidic to basic aminoacid replacements, we hypothesized that similar muta-tions also have the potential to increase transpositionalactivity of the SB transposase. There are altogether 28aspartic acid (D) and glutamic acid (E) residues in the SB

FIG. 4. A region between the DNA-binding and the catalytic domains of the Sleeping Beauty transposase interferes with efficient substrate binding. (A) Mobility

shift experiment using N-123 and N-161 derivatives of the SB transposase and a radiolabeled IR/DR fragment as a probe. N-123 contains the N-terminal 123

amino acids of the transposase, encompassing the DNA-binding domain responsible for binding to two sites within each of the IR/DR repeats. N-161 contains

the N-terminal 161 amino acids of the transposase, encompassing the DNA-binding domain plus a predicted helix. The two retarded bands (arrowheads) in the

N-123 sample represent complexes in which one (faster migrating complex) or both (slower migrating complex) transposase binding sites within the transposon

IR/DR probe are bound by the transposase. The gel shows severe reduction of complex formation by N-161. In the control reaction (C), no protein is added to

the probe. (B) A conserved, predicted helix in Tc1-like transposases between the DNA-binding and the catalytic domains. An amino acid alignment of Tc1-like

transposase segments is shown. The exact amino acid positions at which N-123 and N-161 terminate are indicated. The predicted helical region is boxed, and

small black arrowheads mark those amino acids within the helix in the SB transposase that were replaced by proline residues. The position of the first D residue of

the DDE catalytic triad is indicated. Amino acid sequence alignment, sequence conservation, and secondary structure prediction were done using ClustalW,

BOXSHADE 3.21, and PredictProtein softwares, respectively.



transposase, which are listed in Table 1. We categorizedthese amino acids with respect to their conservation inthe Tc1 family (Table 1). Conserved amino acids likelyplay crucial roles in transposase activity. For example, theDDE residues of the catalytic domain are absolutelyconserved in the transposases and are required for trans-

position [8,36]. Therefore, we did not subject conserved Dor E residues to mutagenesis. The remaining 15 D or Eamino acids were replaced by either a lysine (K) or anarginine (R) residue (Table 1).

Mutations E6K, D10K, D17K, D68K, D86K, E92K,E93K, E158K, D164K, E174K, E216K, and E321R reduced

FIG. 5. Effects of amino acid substitutions on the efficiency of Sleeping Beauty transposition. (A) Effects of single-amino-acid replacements. Shown are the

transpositional efficiencies of 22 single-amino-acid mutants of the transposase relative to the wild-type SB10 transposase (white column). From these, 15 are E to

K/R mutations, 5 are proline mutations in the linker region between the DNA-binding and the catalytic domains (Fig. 4B), and 2 are naturally occurring sequence

variants. The three individual hyperactive mutants identified in this screen are shown as black columns. (B) Effects of hyperactive mutations in combinations.

Transposition was assayed in human HeLa cells, and the activity of wild-type transposase (SB10, white column) is taken as a reference and set to 100%. (C) Effects

of combinations of hyperactive transposases, hyperactive transposons, and HMGB1. Transposition was assayed in human HeLa cells, and the activity of the first-

generation SB system (SB10 plus T/zeo, white column) is taken as a reference and set to 100%.



transposition frequency to barely measurable levels,whereas D140K and D142K reduced transposition toabout 70 and 50%, respectively (Fig. 5A), indicating thatthese amino acids play critical roles in SB transposaseactivity. Importantly, however, transposition activity ofD260K was about 40% higher than that of wild-type SB10(Fig. 5A), demonstrating that an acidic-to-basic change inthis position improves the function of the transposase.

Our third approach to mutagenesis of the SB trans-posase was to introduce amino aids that naturally occurin SB or related transposases. Such an approach hasbeen shown to be useful for the generation of hyper-active versions of SB [18]. We evaluated the effects oftwo amino acid changes in the transposase: R115H andR143C. The R115H substitution was made based on acomparison between SB and the Tdr1 transposase inzebrafish [32]. These two transposable elements repre-sent closely related subfamilies of Tc1-like transposonsin fish genomes and show about 80% identity in trans-posase sequence. Therefore, these two sequences prob-ably represent variants of a transposase that had beenselected for activity in nature. The amino acid residue

in position 115 in the Tdr1 transposase is a histidine,which is expected to preserve the positive charge inthis position of the transposase polypeptide. The sec-ond mutant that we tested, the R143C substitution, is anaturally occurring mutation in the SB transposase,possibly generated at a mutable CpG site in the trans-posase gene. The R143C mutant is also called SB9 andrepresents a particular intermediate version of thetransposase that we obtained during the reconstructionprocess of the SB transposase gene [2]. Both the R115Hand the R143C mutants showed hyperactivity: R115Hby about 60% and R143C by about 25% compared tothe wild-type SB10 transposase (Fig. 5A).

Next, we asked the question whether combinations ofthe D260K, R115H, and R143C hyperactive mutationswould result in an additive or a synergistic effect. Towardthis end, we engineered the three possible doublemutants and a triple mutant. R115H/D260K showed a3.7-fold, R115H/R143C a 3.2-fold, R143C/D260K a 2.6-fold, and the R115H/D260K/R143C combination a 2.3-fold increase in transposition activity compared to thewild-type transposase (Fig. 5B). These results indicate thatthe R115H mutation acts synergistically with both D260Kand R143C. We sought to determine whether incorpora-tion of the previously described T136R/M243Q/VVA253HVR hyperactive mutations (collectively referredto as SB11) [18] would further increase transpositionactivity of our hypertransposing mutants. SB11 showeda 2.3-fold higher activity than wild-type transposase, andthis level of activity remained unchanged when SB11 wascombined with the R115H/D260K mutations (Fig. 5B).

Altogether, our results demonstrate that a mutagenesisapproach to the development of hyperactive transposasesis viable and that the R115H/D260K mutant (hereafterreferred to as SB12) is the most active SB version describedto date.

Combination of Hyperactive Components Results in aFurther Enhancement of Sleeping BeautyTransposition ActivityA reasonable expectation is that combinations of hyper-active transposases with hyperactive transposons result ina further (either additive or synergistic) increase in overalltransposition frequencies. However, this was not the casein a previous study in which the SB11 hyperactive mu-tant was used in combination with an improved trans-poson vector, but was found to show no furtherenhancement [18].

We evaluated our SB12 double mutant in combinationwith two hyperactive transposon vectors. The first, pT/zeo322, is a vector that has only a short segment of DNAoutside the transposon and was previously found to showan approximately twofold increase in the efficiency oftransposition [3]. This effect can probably be attributed tothe improved ability of the two ends of the transposon topair during transposition. The second is based on the T2

TABLE 1: List of aspartic acid and glutamic acid residues inthe Sleeping Beauty transposase

Amino acid residuein SB transposase

Status in other Tc1/marinertransposases

Changemade

E6 Nonconserved KD10 Nonconserved KD17 Nonconserved KD68 Nonconserved KE69 Conserved —D86 Nonconserved KE92 Nonconserved KE93 Nonconserved KD140 Nonconserved KD142 Nonconserved KD153 Conserved —E154 Conserved —E158 Nonconserved KD164 Nonconserved KE174 Nonconserved KD210 Conserved —E216 Nonconserved KD220 Conserved —D244 Conserved —D246 Conserved —D260 Nonconserved KE267 Conserved —D274 Conserved —E279 Conserved —E284 Conserved —E306 Conserved —E307 Conserved —E321 Nonconserved R

Whether these D and E amino acids are conserved in other Tc1-like transposases and the

amino acid replacements that were introduced into the positions of the nonconserved

residues are indicated. The DDE residues of the catalytic triad are shown in bold.



element that has base pair changes in the IRs [23] and hasbeen previously found to show an approximately three-fold enhancement of transposition compared to the wild-type transposon [18]. We modified this vector to have a

short outer distance between the IRs and named it pT2/zeo322. As first-generation components for reference, weused the SB10 transposase and the pT/zeo vector [3]. Asfound before for the SB11 hyperactive transposase [18],

FIG. 6. Effects of different transposase/transposon ratios on the efficiency of Sleeping Beauty transposition. (A) Optimal transposase to transposon ratio is

dependent on the concentration of transfected DNA. 50 ng transposon plasmid (pT/neo) DNA was mixed together with increasing amounts of transposase-

expressing plasmid (pCMV/SB) DNA (50 ng, 1:1; 100 ng, 2:1; 500 ng, 10:1; 1 Ag, 20:1; 1.5 Ag, 30:1), the plasmid mixtures were cotransfected into human HeLa

cells, and the transposition efficiencies were calculated (black columns). Transposition was also measured with the same series of DNA mixtures diluted 5- (gray

columns) or 10-fold (striped columns) before transfection. Transposase vs transposon ratios represent ratios of amounts of plasmid DNAs. 50 ng, 10 ng, and 5 ng

denote the amounts of transposon plasmid DNA per transfection. Transpositional activity measured at the 1:1 ratio for the 50 ng transfection is taken as

reference and set to 100%. (B) Comparison of the transpositional efficiencies of the SB10 (wild-type) and SB12 (hyperactive) transposases at different

transposase/transposon ratios. 500 ng of transposon plasmid DNA was mixed together with either an equal amount (1:1) or 2-, 10-, 24-, or 50-fold diluted

transposase-expressing plasmid DNA (0.5:1, 0.1:1, 0.04:1, 0.02:1, respectively), before transfection into HeLa cells. pCMV-h was used as a filler DNA to keep the

total amount of the transfected DNA constant. Transpositional activity of SB10 measured at the 1:1 ratio is taken as reference and set to 100%.



our SB12 mutant did not act in either an additive or asynergistic way together with the T2-based transposon:the overall efficiency of transposition with this combina-tion was about twofold higher than that of the wild-type(Fig. 5C). However, a combination of the SB12 doublemutant transposase with the pT/zeo322 transposon vec-tor showed an additive effect on transposition and dis-played an about fivefold enhancement in transpositioncompared to wild-type components (Fig. 5C). We nextevaluated the effect of transiently overexpressed HMGB1on transposition rates with the hyperactive transposoncomponents. HMGB1 was previously found to increaseSB transposition in wild-type mammalian cells aboutthreefold [37]. In combination, HMGB1, the SB12 trans-posase, and the pT/zeo322 transposon showed an overeightfold increase in transposition compared to the wild-type SB system (Fig. 5C). This result demonstrates thatthe SB12 hyperactive transposase mutant acts in anadditive manner with a hyperactive transposon and witha rate-limiting cellular host factor in mediating high-efficiency transposition in human cells.

Effects of Absolute and Relative CellularConcentrations of Components of the Sleeping BeautyTransposon System on the Efficiency of TranspositionBecause one transposon molecule contains four trans-posase binding sites, the ideal molar ratio of transposaseto transposon is expected to be 4:1. Indeed, cellularconcentration of the transposase can be a limiting factorof transposition under certain conditions, i.e., increasingexpression of the transposase results in increasing numb-ers of transposition events [3,18]. However, exceedingtransposase expression beyond a certain threshold levelcan have a negative impact on transposition, an effecttermed overproduction inhibition [38]. Consistent withan overproduction inhibition effect, Yant et al. [12] havefound an optimal ratio of 1:25 of SB transposase-express-ing plasmid to transposon donor plasmid in the mouseliver in vivo.

We addressed the issue of molar ratios of the twocomponents of the SB transposon system by measuringtranspositional efficiencies in cells transfected with thetransposase expression plasmid and a transposon donorplasmid in a ratio range of 1:1 to 30:1, but in threedifferent plasmid concentrations. Fig. 6A shows that themore-transposase-more-transposition rule applies at lowconcentrations, but as the transposase concentrationincreases, an inhibitory effect is observed. No inhibitoryeffect was seen at the 30:1 ratio when 5 ng of transposonplasmid DNA was transfected. However, at 10 and 50 ngtransposon DNA, a plateau in transpositional efficiencywas found at the 20:1 and 2:1 ratios, respectively (Fig.6A). Nevertheless, at these concentrations, transfection ofa higher amount of transposase-expressing plasmid thantransposon plasmid was necessary for optimal transposi-tion (Fig. 6A). However, increasing the amount of trans-

fected transposon DNA to 500 ng resulted in a shift inoptimal plasmid ratios (Fig. 6B), indicating that hightransposon concentrations cannot be matched by hightransposase concentrations, possibly due to overproduc-tion inhibition. Under these conditions, dilution of thetransposase plasmid up to 10-fold resulted in more trans-position events, whereas the transposase became limitingupon further dilution (Fig. 6B). Consistent with earlierfindings [18], our observations imply that the optimalplasmid ratios are primarily dependent on how muchDNA is introduced into the cell.

Next, we compared the SB12 hyperactive mutant towild-type SB10 in terms of sensitivity to overproductioninhibition (Fig. 6B). Similar to SB10, transposition bySB12 plateaued at a 1:10 ratio of transposase plasmid totransposon plasmid (Fig. 6B). This result indicates thatthe hyperactive phenotype of SB12 is unlikely to be dueto reduced overproduction inhibition. Importantly, SB12displayed hyperactivity at all of the plasmid ratios tested(Fig. 6B).

These data suggest that regulation of the transposaseby overproduction inhibition sets a limit to transpositionin any given cell and that transposase-to-transposonplasmid ratios need to be carefully optimized for anygiven tissue, either in vitro or in vivo.

DISCUSSION

In this paper, we report a significant enhancement of thetranspositional activity of the Sleeping Beauty transposableelement, a promising vector for nonviral gene transfer invertebrate species. The transposon has two functionalcomponents: the DNA sequences within the terminalinverted repeats of the element and the transposaseprotein. Improvements in the overall activity of thetransposon can be made by manipulating either or bothof these components. Accordingly, we took two experi-mental approaches: (1) modify the structure of the sub-strate transposon by flanking large transgenes withcomplete SB elements, thereby mimicking naturallyoccurring composite elements; (2) generate hypertrans-posing mutants of the transposase by replacing some ofthe nonconserved amino acids.

The Sandwich Sleeping Beauty Element ImprovesTransposition of Long TransgenesWe have shown that the sandwich element has anenhanced capacity to transpose long transgenes (Fig.3B). The structure of the sandwich transposon is some-what similar to that of the bacterial transposons Tn5 andTn10. These elements might have been fortuitously gen-erated by transposition of two insertion sequence ele-ments on both sides of an immobile segment containingantibiotic resistance genes [39,40]. This situation can alsoarise, probably by chance, in other transposition systems,resulting in new, composite, mobile elements. Indeed, a



pair of Paris elements that flank a nonrepetitive sequenceof more than 10 kb in an inverted orientation was shownto be able to transpose in D. virilis [25].

Why does the sandwich vector transpose long trans-genes better than the wild-type SB transposon? Wehave shown earlier that (1) long elements tend totranspose less efficiently than short ones, likely becausethe ends of long elements cannot pair easily duringsynaptic complex formation [3], and (2) the DNA-bend-ing protein HMGB1 plays an important role in SBtransposition likely by aiding the pairing of transposonends [37]. Thus, we suggest that an increase in thenumber of transposase binding sites (from four toeight) in the sandwich transposon can partially rescuesynaptic complex formation of long elements, presum-ably due to the more pronounced action of transpo-sase – transposase interactions and HMGB1 at thetransposon inverted repeats. Artificially made, sand-wich-like Mos1 mariner elements similar to the onesdescribed here for SB have been found to have in-creased mobility in Drosophila [41], suggesting commonunderlying mechanisms in composite transposon mo-bilization in the Tc1/mariner family.

Mutational Analysis of the Sleeping BeautyTransposase and Isolation of Hyperactive MutantsBy using a limited site-directed mutagenesis screen, weidentified hyperactive versions of the SB transposase(Fig. 5A). Three different approaches were undertakenfor the choice of induced mutations: (1) modificationof a linker region that separates the DNA-binding andthe catalytic domains, (2) systematic replacement ofacidic residues with basic amino acids, and (3) substi-tution for amino acids that had been selected in nature.The predicted helix spanning the region between theN-terminal DNA-binding domain and the catalytic do-main is conserved in the Tc1 family (Fig. 4B) and is notpart of the DNA-binding domain [2]. When present inrecombinant transposase preparations, this region inter-feres with binding to the transposon inverted repeats(Fig. 4A). We reasoned that disruption of the localstructure at this region might result in a conformation-al change of the transposase that is more favorable forDNA binding. Proline is a secondary structure breakerin proteins and it has been widely used to modify theconformation of proteins. A hyperactive Tn5 transpo-sase mutant was generated by introducing a prolineresidue that is thought to interrupt the interferencebetween the N-terminal and the C-terminal regions ofthe transposase [35]. Unfortunately, the mutants thatwe generated all showed severely impaired transposi-tion (Fig. 5A), indicating the functional importance ofthis helix in SB transposition. Future work should bedirected to random mutagenesis of the helix in thehope that less drastic changes could result in hyperac-tive phenotypes.

The rationale behind the change of all nonconservedacidic amino acid residues to basic amino acids is that inseveral transposition systems, including Tn5 [26,28],Tn10 [30]. and Himar1 [31], some hyperactive mutationsfall into this class. Acidic-to-basic amino acid changesmight eliminate (or at least reduce) the unfavorablecharge–charge interaction between the acidic amino acidresidues and the negatively charged phosphate backboneof the transposon (or target) DNA [28] or might overcomethe self-inhibiting properties of transposase [26]. Most ofthe mutations that we introduced into the SB transposaseresulted in a decrease in the efficiency of transposition(Fig. 5A), suggesting very little functional redundancy inthe transposase sequence. A marked sensitivity of trans-posase to mutations was previously noted for the Mos1mariner element [42]. Nevertheless, one of the substitu-tions, the D260K mutation, produced a hyperactive phe-notype (Fig. 5A). The aspartic acid in position 260 iseither lysine or arginine in other Tc1-like transposases(Fig. 7), suggesting that lysine and arginine can betterfunction in that sequence context. It is possible that aparticular version of fish Tc1-like transposases did con-tain K or R at position 260, but this amino acid gotreplaced at some point during transposase evolution,because it is functionally nonessential for the transpo-sase. It is important to note that four hyperactive muta-tions reported from Tc1/mariner elements are locatedwithin the same 8-amino-acid segment in the catalyticdomains of these transposases (Fig. 7). The D260K muta-tion (this work) and the V253H and A255R mutations[18] in Sleeping Beauty, and the H267R mutation inHimar1 [31], all map to the same region just precedingthe E/D residue of the DDE (DDD in mariner elements)catalytic triad (Fig. 7). It is therefore possible that thesemutations result in a slight conformational change that ismore favorable for catalysis. Because three of these fourmutations are K and R replacements (Fig. 7), the localshift to positive charge might enhance target DNA cap-ture, a function likely encoded in the catalytic domain[43,44]. Further biochemical work will be required tosubstantiate either of these hypotheses.

The D260K mutation acts synergistically with twoother, naturally occurring mutations, R115H andR143C (Fig. 5B). The R115H/D260K and R115H/R143Cdouble mutants exhibited about 3.7- and 3.2-fold in-crease in transposition activity over wild-type transpo-sase, respectively (Fig. 5B). It seems that the R115Hmutation is important in the double mutants since theR143C/D260K mutant showed only about 2.6-fold in-crease in activity (Fig. 5B). We found that hyperactivityof the R115H/D260K mutant (referred to as SB12 bykeeping the convention of naming versions of the SBtransposase) cannot be attributed to a reduction in over-production inhibition (Fig. 6B). Importantly, SB12 dis-played additive effects with a hyperactive transposonvector (Fig. 5C). Transposition of this hyperactive system



is further enhanced by transient overexpression ofHMGB1, a cellular cofactor of SB transposition [37](Fig. 5C). The collective effect of these components isan approximately eightfold increase in transposition,compared to the first-generation SB system (Fig. 5C). Inaddition to relative efficiencies of transposition, it isuseful to calculate the absolute numbers of transformantcells that can be generated with transposition in a giventransfected cell population. For this, we cotransfected aGFP expression construct together with the transposoncomponents into human HeLa cells and normalizedtransposition rates with transfection efficiencies, basedon GFP fluorescence (data not shown). We found thatabout 2% of cells that had taken up transposon DNA willundergo a transposition event using the first-generationSB system, whereas stable transgenesis rates are about10–15% using combinations of the hyperactive transpo-son/transposase components. Transposition can be fur-ther optimized by systematic adjustment of transposaseand transposon concentrations in transfected cells, be-cause different ratios of the transposase expression andtransposon donor plasmids can greatly influence trans-position efficiencies (Fig. 6).

Although Sleeping Beauty is an element reconstructedfrom transposon fossils, it most likely represents a trans-poson that was once active in fish genomes. The fact thathyperactive versions of the SB transposase can be gener-ated implies that this transposase has not been selectedfor the highest possible activity in nature. This is becausetransposition can potentially endanger the survival of thehost organism and, consequently, that of the transpos-able element. The overall activity of SB for in vivo genetransfer can be improved by matching the transposonwith highly efficient DNA delivery technologies and byincreasing its activity in the cell. In this paper we provid-

ed evidence that the latter strategy is clearly promisingfor the development of more efficient transposon-basedgene vectors.

MATERIALS AND METHODS

Plasmids. The CA ! GC change in the right IR was induced in pT

and pT/neo plasmids using the primers FTC-3 [2] and SB-SAND (5V-

TTGCATCTAGAGCGGCCGCGTTGAAGTCGGAAGTTTACATACACCT-

TTAGCC-3V) for pT and PR-Neo (5V-CCTTGCGCAGCTGTGCTCGACG-

3V) and SBSAND for pT/neo. The SB-SAND primer introduces XbaI (bold

italic) and NotI (bold) restriction sites. The mutated rIR was subcloned

in pT and pT/neo to yield pT* and pT*/neo. pEGFPC1 (Clontech) was

linearized with AseI, Klenow-filled, and ligated with NotI-digested,

Klenow-filled pT*, yielding the cloning intermediate pT*pEGFPC1.

The same fragment was also cloned into the BlpI site in pT to yield

pT6.2. The complete T* transposon was moved in an EcoRI/XbaI

fragment into pUC18 and subsequently cloned in an XbaI/ScaI frag-

ment into pT*pEGFPC1 to yield the sandwich transposon pT/SA7.7,

which has the 4.7kb EGFPC1 fragment between two T* transposons in

an inverted orientation. pT/SA7.7 was linearized with BglII, Klenow-

filled, and ligated with a 4.5kb PstI fragment of pCMVh (Clontech)

containing a CMVpromoterdriven lacZ gene, to yield pT/SA12.2.

Site-directed mutagenesis of the transposase gene was done by PCR

using pCMV-SB as a template. Primer sequences are available from the

authors upon request. All mutations were confirmed by sequencing.

pT/neo deletion derivatives were generated by PCR using primers that

generate an SpeI site. Primers for pT/neo-SpeI were 5V-AGTCCTTGAAATA-

CATCCACAGGTACAG-3Vand 5V-AGTCTGTGGAATGTGTGTCAGTTAGG-

3V. Primers for pT/neo-SpeI/D112 were 5V-AGTCTGTGG-AATGTGTGT-

CAGTTAGG-3V and 5V-AGTTTAAAGGCACAGTCAACTTAGTG-3V. Primers

for pT/neo-SpeI/D46 were 5V-AGTTTAAAGGCACAGTCAACTTAGTG-3Vand

5V-AGTCTGATA-GACTTATTGACATCATTTGAG-3V. Primers for pT/neo-

SpeI/D66 were 5V-AGTCTGTGGAATGTGTGTCAGTTAGG-3V and

5V-AGTAAGCTTCTAAAGCCATGACATCATTTTC-3V. Primers for pT/neo-

SpeI/D112-mut were 5V-AGTTTAAAGGCACAGTCAACTTAGTG-3V and

5V-AGTCACGTTCATGAGTCAACTTAGTGTATGTAAAC-3V. The PCR prod-

ucts were circularized after T4-kinase treatment by ligation and trans-

formed in Escherichia coli (DH5a). Clones were verified by SpeI digestion.

Bacterial expression vectors were made in pET21a (Novagen), encoding

a C-terminal histidine tag. pET21a/N-123 was described earlier [2]. pET21a/

FIG. 7. Locations of hyperactive mutations. Amino acid alignment of transposase segments of Tc1-like transposons and that of the Himar1 mariner element

highlights a region where several hyperactive mutations are located. The alignment shows that D260 of SB is not conserved: several Tc1-like transposases contain

lysine or arginine in this position. The E residue (D in Himar1) of the DDE catalytic triad is indicated.



N-161 was made by cutting a pET21a-derived plasmid containing the full-

length transposase gene with MscI/NotI and recircularizing the plasmid.

Cell culture and transfections. HeLa cells were maintained in DMEM

containing 10% fetal bovine serum. Transposition assays were done as

described [2]. If not stated otherwise, 105 cells were transfected with 90 ng

each of the transposon donor plasmid and the transposase-expressing

helper plasmid using Fugene6 transfection reagent (Roche). Two days

posttransfection, the cells were replated and selected in 1.4 mg/ml G418.

After 2 weeks of selection, the resistant colonies were stained and counted.

Recombinant protein expression and purification. Induction of His-

tagged, recombinant protein expression was in E. coli strain BL21(DE3)

(Novagen) by the addition of 0.4 mM IPTG at 0.5 OD at 600 nm and

continued for 2.5 h at 30jC. Cells were sonicated in H-buffer [25 mM

Hepes (pH 7.5), 15% glycerol, 0.25% Tween 20] containing 2 mM h-

mercaptoethanol, 1 M NaCl, and 1� Complete (Boehringer Mannheim),

and 20 mM imidazole (pH 8.0) was added to the soluble fraction before it

was mixed with Ni2+ –NTA resin (Qiagen) according to the recommenda-

tions of the manufacturer. The resin was washed with sonication buffer

containing 30% glycerol and 50 mM imidazole, and bound proteins were

eluted with sonication buffer containing 300 mM imidazole and dialyzed

overnight at 4jC against sonication buffer without imidazole.

Electrophoretic mobility shift assay. The probes containing either the

left or the right IR of SB were end-labeled using [a-32P]dATP and Klenow.

Nucleoprotein complexes were formed in 20 mM Hepes (pH 7.5), 0.1 mM

EDTA, 0.1 mg/ml BSA, 150 mM NaCl, 1 mM DTT in a total volume of 10

Al. Reactions contained labeled probe, 1 Ag poly(dI –dC), 100 pg labeled

fragment, and 1 pmol protein. After 15 min incubation on ice, 5 Al of

loading dye containing 50% glycerol and bromophenol blue was added,

and the samples were loaded onto a 4% polyacrylamide gel.

ACKNOWLEDGMENTS

We thank E. Stuwe and A. Katzer for their technical assistance and members of

the Transposition Group at the MDC for critically reading the manuscript. This

work was supported by EU Grant QLG2-CT-2000-00821.

RECEIVED FOR PUBLICATION SEPTEMBER 23, 2003; ACCEPTED NOVEMBER

29, 2003.

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Documents

Development of Hyperactive Sleeping Beauty Transposon