Endothelial targeting of the Sleeping Beauty transposon within lung

Endothelial Targeting of the Sleeping BeautyTransposon within Lung

Li Liu,1 Sonia Sanz,1 Arnold D. Heggestad,1 Vijay Antharam,1

Lucia Notterpek,2 and Bradley S. Fletcher*,1,3

1 Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610, USA2 Department of Neuroscience, University of Florida College of Medicine, Gainesville, FL 32610, USA

3 Medical Research Service, Department of Veteran Affairs Medical Center, Gainesville, FL, USA

*To whom correspondence and reprint requests should be addressed at the Department of Pharmacology and Therapeutics,

University of Florida College of Medicine, Box 100267, 1600 SW Archer Road, Gainesville, FL 32610-0267, USA.

Fax: (352) 392-9696. E-mail: [email protected].

Available online 13 May 2004

Endothelial cells have complex roles in the pathophysiology of vascular and heart disease and areincreasingly being recognized as targets for gene therapy. The intravenous administration ofplasmid DNA complexed to lipid tends to target transfection of endothelial cells within the lung;however, expression from the transgene remains transient. Here we utilize the integratingcapability of the Sleeping Beauty (SB) transposon for durable gene transfer within lung endothelia.To restrict expression of the transgene, an endothelial cell-specific promoter, endothelin-1, wasplaced within the transposon. Further refinements to the transposon increased in vitrotransposition efficiency by 3.6-fold. Utilizing this optimized transposon we evaluated theexpression of two reporter molecules, secreted alkaline phosphatase (SEAP) and intracellularGFP, following administration of DNA–polyethylenimine complexes to mice. Long-termexpression (>2 months) of SEAP occurred only with cotransfection of adequate amounts oftransposase. Localization studies using the GFP reporter, at 3 days and 6 weeks postinjection,demonstrated that the majority of transgene-expressing cells were of endothelial origin, whilethe second most abundant cell type was type II pneumocyte. These results suggest that the SBtransposon can be adapted to target particular cell types, in this case, endothelial cells. Such anapproach may be useful for gene therapy paradigms involving the long-term modulation ofvascular and endothelial cell biology.

Key Words: Sleeping Beauty, transposon, endothelial cell, gene therapy, polyethylenimine,nonviral vector

INTRODUCTION

Efficient and sustained transfer of genetic material is thecommon goal of most gene therapy approaches. Whileviral-based vector systems, including adenovirus, adeno-associated virus, retrovirus, and lentivirus, have shownconsiderable promise, there are limitations and/or poten-tial safety concerns for these delivery vehicles [1,2]. Non-viral gene-transfer approaches have recently gainedincreasing attention as they provide certain advantagesover viral systems [3]. These include the ability to accom-modate large amounts of genetic material, the lack ofimmune response to the delivery vehicle, reasonable safe-ty profiles, simplified production, and low manufacturingcosts. However, a major obstacle to nonviral approaches

has been the lack of persistent gene expression necessaryfor a sustained therapeutic affect. This loss of activityfollowing nonviral gene transfer is attributed to the lackof genomic integration of the administered genetic mate-rial. Plasmid-based transposons with activity in vertebratecells are a mechanism by which one can facilitate genomicintegration and thus overcome this significant obstacle.

Transposons are mobile genetic elements found in avariety of species including bacteria, plants, and insects.The Tc1/mariner-like Sleeping Beauty (SB) transposase wasfunctionally resurrected from teleost fish sequences [4].Transposition of SB occurs by a ‘‘cut-and-paste’’ mecha-nism, inserting a transposon flanked by two-invertedrepeat/direct repeat (IR/DR) elements into a TA dinucle-otide sequence found within plasmid or genomic DNA

MOLECULAR THERAPY Vol. 10, No. 1, July 2004 97Copyright B The American Society of Gene Therapy

1525-0016/$30.00

doi:10.1016/j.ymthe.2004.04.006 ARTICLE

targets. This activity requires only the expression of thetransposase and can function in a wide variety of verte-brate species, including cell lines derived from human,murine, bovine, and fish origin [5]. SB is the most activetransposon within mammalian cells [6]. Given its en-hanced activity, SB has recently become a useful toolfor insertional mutagenesis [6,7] and gene therapy [8–10]. A recent review describes the strengths and limita-tions of transposon-based gene delivery [11].

Since the initial characterization of SB, several studieshave investigated the determinants that influence theefficiency of transposition within mammalian cells invitro [5,12–14]. Alterations to the transposon haveyielded important information concerning general rulesof SB transposition and impact the use of SB as a gene-delivery vehicle. These reports show that shortening theinner or outer distances between IR/DR elementsincreases the efficiency of transposition [5]. Furthermore,changes within the right-side IR/DR element or supple-mentation of an additional TA dinucleotide to the out-side ends of the transposon can enhance transposition[12,13]. Modifications to the transposase to generatehyperactive mutants are also actively being pursued[14,15]. Despite these advances in understanding SBtransposition, there are no published reports on the useof such enhanced transposons or transposases in vivo.

Here we describe experiments to optimize a SB-basedvector system for targeted delivery of endothelial cells invivo. Endothelial cells are ideal targets for gene therapygiven their ability to regulate vascular tone and hyperten-sion as well as modulating local thrombosis, arterioscle-rosis, and neointimal hyperplasia. To restrict transgeneexpression within endothelial cells, the promoter regionof the endothelin-1 gene (ET-1; approved gene symbolEDN1) is utilized. Modifications to the transposon thatdemonstrate increased transposition efficiency in vitro areincorporated. Following injection into mice, we observelong-term (greater than 2 months) expression of a secret-ed alkaline phosphatase reporter (SEAP) gene within theserum that is dependent upon the coadministration oftransposase. Localization studies reveal that both endo-thelial cells and type II pneumocytes within the lungexpress the reporter gene GFP. This system thereby pro-vides an approach for targeting long-term expressionwithin endothelial cells in vivo and has potential applica-bility for a variety of gene therapy paradigms.

RESULTS

Generation of Transposon Constructs and Evaluationof Transposition EfficiencySeveral factors that affect the transposition efficiency ofthe SB transposon have been identified [5,12–14,16]. Asignificant contributing factor is the amount of DNAboth inside and outside of the IR/DR elements [5].Changes within the IR/DR elements themselves, as well

as the composition of the DNA surrounding the ends ofthe IR/DR elements, can also influence transposition[13,16]. Utilizing these findings, we have designed anoptimized SB-based vector system for gene delivery toendothelial cells. To restrict expression within endothe-lial cells, we have placed the ET-1 promoter between theIR/DR elements (designated pE in Fig. 1A). The ET-1promoter has been shown to provide relatively specificexpression within endothelial cells [17]. We conductedfurther modifications of this vector system to increase thetransposition efficiency within mammalian cells. Thesemodifications included changes to the right-side IR/DRelement (pEM), combined with shortening the inner IR/DR distance (pMSF) or shortening both the inner and theouter IR/DR distances (pMSZ) (Fig. 1A).

We evaluated the transposition efficiency of theseconstructs in an in vitro assay using HeLa cells (Fig. 1B).For these experiments, we employed a humanized ver-sion of the SB10 transposase driven by a CMV promoter(pCMV-HSB) identical to that of the parental plasmidpCMV-SB. Comparisons between these two plasmids us-ing the reporter pT-SVNeo suggest that they have approx-imately equal activity (Fig. 1B). Modifications to theright-side IR/DR element (pEM) as described by Cuiet al. [13] resulted in only a modest improvement (1.35-fold) in transposition efficiency relative to plasmid pE.As expected, shortening the inner (pMSF) or both innerand outer IR/DR distances (pMSZ) enhanced transposi-tion by 1.3- and 1.8-fold, respectively, relative to pEM.The most active transposon (pMSZ) combines the right-side IR/DR element changes with both of the shortenedinner and outer IR/DR distances, resulting in a 3.6-foldimprovement over the parental plasmids pT-SVNeo andpCMV-SB. These data support the hypothesis thatsmaller transposons are more efficient at transpositionthan larger ones. Furthermore, when combined, thesesmall changes appear to be additive, thus resulting in amore efficient transposon.

The SB transposon has been shown to have varyinglevels of transposition activity depending upon the celltype involved, even within the same organism [5]. Toassess if endothelial cells were conducive to SB transpo-sition, we performed in vitro transposition assays in hu-man umbilical vein endothelial cells (HUVECs).Following transient transfection with the neomycin-expressing transposon pMSZ-neo, in the presence orabsence of the transposase-expressing plasmid (pTRUF-HSB), we selected cells for neomycin resistance. Thisparticular transposase expression plasmid was chosenbased on the observation that this promoter was stronglyactive within endothelial cells. These studies (Fig. 1C)demonstrate that HUVECs have a higher propensity toform spontaneous neomycin-resistant colonies followingtransfection of the pMSZ-neo reporter alone (f1 in 75transfected cells became resistant). However, the additionof transposase increased the frequency of neomycin-re-

MOLECULAR THERAPY Vol. 10, No. 1, July 200498Copyright B The American Society of Gene Therapy

ARTICLE doi:10.1016/j.ymthe.2004.04.006

sistant colonies by 6.5-fold. Based on the transfectionefficiency of 15%, we estimate that f9% of transfectedcells become long-term stable expressers. Thus, endothe-lial cells of human origin are conducive to SB transposi-tion, albeit not as efficiently as HeLa cells.

Evaluation of Endothelin-1 Promoter Construct inTissue Culture CellsIn an effort to restrict expression of the transgene toendothelial cells in vivo, we designed the transposon tocontain the ET-1 promoter. Initially characterized byInoue et al. [18], the ET-1 promoter was later shown tocontain an upstream regulatory enhancer that was re-sponsible for high-level endothelial cell-specific expres-sion [19]. More recent evaluation by Cho et al. [17]demonstrated robust expression in endothelial cells usinga luciferase reporter construct compared to a CMV-drivenreporter. Our particular human ET-1 promoter constructcontains sequences from �251 to +174 relative to thetranscription start site. In addition, the murine endothe-lial enhancer region (�364 to �320) was multimerizedfour times and placed upstream of the promoter. Toevaluate the specificity of this promoter, we investigatedthe relative fluorescence levels in several different celllines following transient transfection of either a CMV-driven (pGreenLantern) or an ET-1-driven GFP expressionconstruct. We chose to evaluate the expression profileswithin cell types that are present in blood vessels, includ-

ing endothelial and smooth muscle cells, as well as fibro-blasts. As illustrated in Fig. 2, the ET-1 promoter is notentirely endothelial cell specific, as low-level GFP expres-sion is seen in fibroblasts and smooth muscle cells. Evenwith transfection efficiencies of f50% within the 3T3 andA7R5 cells, less than 25% of ET-1-transfected cells wereGFP positive and the mean fluorescence intensities (MFI)of the GFP-positive populations were lower than those ofthe CMV-GFP-transfected cells. In contrast, within endo-thelial cells (HUVECs) the ET-1 promoter expressed highlevels of GFP, even higher than that observed for theCMV-driven GFP (MFI 779 versus 641). These studiesshow that the ET-1 promoter provides robust expressionof transgenes within endothelial cells; however, this ex-pression may not be exclusive to those cells.

SEAP Expression Following Transposon/TransposaseCo-injectionTo deliver the SB transposons to lung endothelia wechose to utilize the commercially available cationic poly-mer polyethylenimine (PEI), which has previously beenshown to transfect lung with high efficiency [20–23].Five-week-old C57Bl/6–SCID mice received a total of 60Ag of plasmid DNA using a fixed amount of transposon(30 Ag pMSZ-SEAP) and variable amounts of transposase(0, 2, 15, and 30 Ag pTRUF-HSB). The reporter geneexpressed by the transposon in these studies encodes asecreted form of alkaline phosphatase that is heat stable

FIG. 1. Enhanced transposition of the ET-1-

driven transposons. (A) Schematic represen-

tation of the various ET-1 transposon con-

structs. Inverted repeat/direct repeat

elements (IR/DR) flank the neomycin ex-

pression cassettes driven by the indicated

promoters. The right-side IR/DR element

has been modified (gray) in plasmids pEM,

pMSF, and pMSZ. Endo, ET-1; pA, poly(A)

sequence. (B) Transposition assays of the

transposon constructs in HeLa cells. The y

axis is fold transposition relative to the

cotransfection of the constructs pT-SVNeo

and pCMV-SB. In these studies, equal molar

ratios of transposon/transposase were trans-

fected (0.46 nmol) to allow comparisons

between the constructs. (C) Transposition

assay within HUVE cells. The number of

actual neomycin-resistant colonies is re-

ported on the y axis. The data plotted for

B and C represent the means and standard

deviations of three independent experi-

ments performed in duplicate.



[24,25]. This allows for the simple assessment of alkalinephosphatase activity within heat-inactivated serum as ameasure of sustained gene expression. We obtained se-rum samples from animals in each experimental group atmultiple time points (1, 2, 3, 5, 7, 14, 28, 42, 56, and 70days) following injection of the DNA–PEI complexes andanalyzed them for SEAP activity. We observed peak ex-pression of SEAP protein (f2600 ng/ml) from animals inall experimental groups at f48 h postinjection (Fig. 3). Atthe 7-day time point, there was a significant reduction inSEAP activity in the experimental groups that received no(Group 1) or low (Group 4) amounts of transposase. Incontrast, SEAP activity remained stable, at about f200–300 ng/ml, in animals from the groups that contained 30(Group 2) or 15 Ag (Group 3) of transposase. These levelsof SEAP activity remained stable for Groups 2 and 3throughout the remainder of the study, out to day +70following injection. In comparison, SEAP protein levels

for Groups 1 and 4 slowly fell to background levels by day56. These data support the hypothesis that addition ofthe transposase facilitates genomic integration and thuslong-term gene expression of the reporter transposon.

Localization of GFP Expression in Lung TissueTo determine the tissue distribution and cellular localiza-tion of transgene expression following PEI-mediated de-livery of the ET-1-driven transposons, we co-injected aseparate group of animals with the GFP-encoding trans-poson (pMSZ-GFP) and the transposase expression plas-mid (pTRUF-HSB). We obtained perfusion-fixed tissuesfrom several organs, including lung, heart, liver, spleen,and kidney, from control animals and 3 days and 6 weeksafter DNA injection. We evaluated GFP fluorescence bymicroscopy of cryosections from each of these tissues.Compared to control samples (Fig. 4A), we observed brightGFP fluorescence in some cells of the lung from mice that

FIG. 2. Evaluation of ET-1 promoter specificity in

various cell lines. Plasmids expressing GFP driven

by either the CMV or the ET-1 promoter (Endo)

were transiently transfected into the indicated cell

types. FACS analysis was performed at 48 h

posttransfection and the percentage of GFP-pos-

itive cells (% in right upper quadrant), as well as

the mean fluorescence intensity (MFI) of the GFP-

positive populations, is reported.



received the GFP transposon at both the 3-day (Fig. 4B)and the 6-week time points (Fig. 4C). Arrows (in Figs. 4Band 4C) illustrate the GFP-positive cells. Besides rare GFP-positive cells within the liver at the 3-day time point, theother tissues examined had no significant GFP expressionat either time point (data not shown). As seen previouslywith PEI-mediated gene delivery, transfected cells werewidely distributed within the alveolar spaces of the lung[20]. GFP fluorescence was not observed in large bloodvessels or bronchioles. To assess transfection (early timepoint) and gene transfer (late time point) efficiencies, wecalculated the percentage of GFP-positive cells within thelung at both the 3-day and the 6-week time points (Fig.4H). These studies show initial transfection rates of f22%,followed by long-term gene transfer rates of f2–3%.These results correlate with the transposition efficiencyof the enhanced SB transposons at f10% shown in Fig. 1B.

To characterize the cell specificity of GFP expression,we performed immunolabeling studies with antibodies toendothelial cells as well as type II pneumocytes. Usingrabbit polyclonal antibodies to von Willebrand factor (anendothelial marker) (Figs. 4D and 4F) and surfactantprotein A (a type II pneumocyte marker) (Figs. 4E and4G), we found colocalization of GFP with both of thesemarkers (Figs. 4D–4G). Arrowheads in each of the figuresdemonstrate colocalization of GFP with the specificmarker, whereas arrows denote cells that are GFP positive,but do not colocalize with the given marker. By quanti-fying the frequency of GFP colocalization with each ofthese markers, we observed that the majority of GFP-positive cells (f50%) are of endothelial origin at both the

3-day and the 6-week time points (Fig. 4I). Type IIpneumocytes are the second most abundant cell typeand account for f34% of the GFP-positive cells. Theseresults support the notion that the ET-1 promoter is notentirely endothelial specific, yet does favor expressionwithin endothelial cells.

DISCUSSION

Gene delivery for the treatment of heritable disorders orother chronic conditions requires long-term and high-level expression of the therapeutic molecules. Viral-basedvectors have been the favored method of gene delivery asthey offer high transduction efficiencies and mechanismsfor integration and long-term expression [1,2]. Non-viral-based gene delivery has recently received increased atten-tion due to its improved safety profile and diminishedproduction costs; however, lack of long-term transgeneexpression has been a substantial drawback. This limita-tion can be overcome through the use of mechanisms tofacilitate genomic integration [8,26]. The Tc1-like SleepingBeauty transposon is the most promising of these non-viral-based approaches as it is the most efficient at stablyintegrating therapeutic transgenes [6]. To date, the in vivouse of SB-based gene delivery has targeted mainly theliver through the use of hydrodynamic injection of nakedDNA [8,9]. However, SB-based gene transfer to the lunghas recently been described using DNA–PEI complexes,which resulted mainly in transposition within type IIpneumocytes [10]. As intravascular introduction ofDNA–lipid complexes tends to transfect the endothelialand epithelial cells of the lung, we chose to develop a SB-based vector system that is designed to target expressionwithin endothelial cells. Heart and vascular diseaseremains a significant cause of morbidity and mortality,thus the ability to target genetic material to sites thatinfluence the pathophysiology of these disorders is ofmajor interest. Gene therapy of endothelial cells could beutilized to treat a number of conditions, including theproduction of therapeutic proteins for release within theserum, the modulation of vascular tone and hyperten-sion, and the regulation of vascular platelet aggregationand thrombosis, as well as the ability to influence arterio-sclerosis and intimal hyperplasia.

Factors influencing the efficiency of SB transpositionare not well understood and may vary depending uponthe cell type or species studied [5]. Within HeLa cells,both the inner and the outer distance between the IR/DRelements have been shown to influence transposition[5,14]. Based on these conclusions, we designed a set ofoptimized transposons for use in gene delivery and eval-uated their transposition efficiency within HeLa cells.Our results confirm the previous findings and suggestthat the most favorable transposons for SB-based genetherapy should be made as small as possible. Modifica-tions to the IR/DR elements within the transposon can

FIG. 3. Long-term SEAP activity in mice following delivery of SB transposons.

C57Bl/6 –SCID mice were randomly classified into four groups (n = 5 per

group) and received 30 Ag of pMSZ-SEAP transposon with various amounts of

pTRUF-HSB, 0 (Group 1), 30 (Group 2), 15 (Group 3), and 2 Ag (Group 4), via

tail vein injection. Plasmid pcDNA3.1 was used as nonspecific filler to maintain

the total DNA at 60 Ag. Serum was obtained by tail bleeding at the times

indicated (days +1, 2, 3, 5, 7, and 14 and every 2 weeks thereafter up to day

+70) and assayed for SEAP activity. The amount of SEAP protein for each

sample was quantified by comparison to a standard curve and reported as ng/

ml. The data represent the mean SEAP protein levels F the standard error of

measurement.



also enhance transposition [12,13]. The refinements tothe right-side IR/DR element within our vectors didslightly improve transposition efficiency, but not to theextent described previously [13]. Last, the optimal ratio oftransposon to transposase may vary depending upon thecell type or tissues transfected. In liver-targeted SB genedelivery significantly reduced transgene expression wasobserved when the transposon/transposase ratio was near1:1, and optimal expression was seen at a 25:1 ratio [8].The reason for this effect was presumed to be overpro-duction inhibition, which has been demonstrated in vitroby altering transposon/transposase ratios [14,15]. In con-trast, our studies, as well as those by Belur et al., suggest

that for lung tissue, transgene expression is highest withlower transposon/transposase ratios (1:1 or 2:1) [10]. Thereasons for these differences are not clear, yet theysuggest that SB-based gene delivery of specific targettissues will need to be optimized for factors such as sizeof the transposon, addition of cis-acting transposases, andoptimal transposon/transposase ratios.

SB-based gene delivery allows for long-term expressionof transgenes by facilitating the genomic integration ofthe transposon. Our results show that only in the pres-ence of transposase is long term-expression of SEAPprotein maintained. At the longest time point (70 days),the level of SEAP expression (f200 ng/ml) is still nearly

FIG. 4. GFP expression and colocalization

studies in the mouse lung following SB-

mediated gene delivery. Animals received

60 Ag of plasmid DNA (40 Ag of pMSZ-GFP

and 20 Ag of pTRUF-HSB) via tail vein

injection and were sacrificed at 3 days or 6

weeks postinjection. Lungs were fixed and

sectioned as described under Materials and

Methods. Sections from (A) control animals

and (B) 3 days or (C) 6 weeks postinjection

were evaluated for GFP expression. Parallel

sections were stained with von Willebrand

factor (vWF) (a marker of endothelial cells) or

surfactant protein A (SP-A) (a marker of type II

pneumocytes) antibodies. The GFP and (D, F)

anti-vWF or (E, G) anti-SP-A images were

merged to visualize cells expressing both

molecules. The nuclei were counterstained

with Hoechst dye for total cell number

quantification. All images were at 60�original magnification. (H) Percentage of

GFP-positive cells was quantified at both the

3-day and the 6-week time points and is

expressed as percentage of GFP-positive cells

F SD. (I) Percentage of transgene expression

colocalizing with either endothelial cells or

type II pneumocytes. The data are expressed

as percentage of GFP-positive cells F SD.



100-fold higher than in controls. Several prior publica-tions have confirmed the efficiency of long-term expres-sion to be about f2 to 5% [8–10]. Based upon our GFPlocalization studies within the lung, we observe a similarlevel of long-term gene expression at f2–3%. For thestudies presented here, linear PEI was utilized as the DNA-complexing agent. Although efficient in its delivery tothe lung, the alveolar region, primarily pneumocytes,tends to be the primary site of transfection and notendothelial cells [27]. By incorporation of a promoterthat is relatively endothelial cell specific, we were ableto influence this expression pattern to increase the num-ber of transgene-positive endothelial cells. Based on theliterature, this is the first demonstration of cell-specifictargeting using in vivo delivery of SB transposons.

Targeted expression within endothelial cells followinggene delivery can occur through methods that modulatewhere gene delivery occurs or through the use of endo-thelial-specific promoters. Several lipid-based formula-tions that tend to target gene delivery to endothelialcells have been evaluated [28]. These approaches havebeen further modified by the use of antibodies to endo-thelial cell receptors [29,30] or RGD-like motifs thatdirect DNA–lipid complexes toward endothelial cells[31]. These gene delivery approaches could be used inconjunction with cell-type-specific promoters to regulatestrongly where transgene expression occurs. Our findingsshow that while the ET-1 promoter is not entirely endo-thelial cell specific, it does promote strong transcriptionwithin endothelial cells in vitro. The in vivo localizationstudies, which show mainly endothelial cell-specific ex-pression, further support the hypothesis that the use of acell type-specific promoter within the SB transposon canregulate expression within tissues. This observation hasalso been made using a transposon harboring the g-crystallin promoter, which directed expression withinthe eyes of transgenic fish [32]. In these examples, thetransgene within the transposon is transcriptionally reg-ulated in certain cell types. We envision that cell-specificregulation of transposase activity may also be used totarget the cell types in which gene integration occurs. Bycombining techniques to regulate both the types of cellsthat are transfected and the types of cells able to expressthe transposase and transgene, researchers will furtherincrease the safety and efficacy of SB-based gene therapy.

MATERIALS AND METHODS

Plasmid construction. The construction of the optimized transposons

was generated through multiple cloning steps involving several different

plasmids. The IR/DR elements from plasmid pT-SVNeo (a gift from Dr.

Perry Hackett, University of Minnesota) were first cloned into pBluescript

II KS to generate pIR2X. The left IR/DR was removed with KpnI and

BamHI, blunted, and cloned into the SmaI site, while the right IR/DR was

removed with EcoRI and SalI and cloned into the EcoRI –SalI sites of

pBluescript. The unique KpnI and NotI sites within pIR2X were destroyed

and an oligonucleotide linker containing restriction sites for NarI and

KpnI was cloned between the IR/DR elements using the PstI – EcoRI sites.

The unique NarI –KpnI sites were then used to clone the ET-1 expression

cassette from a derivative of plasmid pGS1393. The plasmid pGS1393 (a

gift from Dr. Sean Sullivan, University of Florida) contains a multi-

merized (4�) region of the ET-1 enhancer, the ET-1 promoter and first

exon (f420 bp), a short synthetic intron, a gene, and the human growth

hormone poly(A) signal. The gene within this plasmid was removed

using the NcoI –XbaI sites, and a NotI-containing linker was cloned in its

place. PCR amplification of the neomycin-resistance gene was performed

using primers that contained NotI sites and the PCR fragment was cloned

into the NotI linker. The NarI –KpnI fragment (containing the entire

expression cassette) of the Neo-containing pGS1393 was cloned into

pIR2X, generating pE-neo. To make modifications to the right-side IR/DR

element, the KpnI –SalI fragment from pE-neo was removed and cloned

into pUC19. Using PCR techniques and PCR sewing, mutations were

made identical to those described by Cui et al. [13]. This modified IR/DR

element was recloned back into the KpnI –SalI sites of pE-Neo, generating

pEM-neo. To shorten the inner distance between the IR/DR elements,

PCR was performed using oligonucleotides (containing HindIII-bearing

ends) that removed unwanted sequences from the 5V and 3V ends of the

ET-1 expression cassette. This PCR fragment was cloned into the HindIII

sites (present within each IR/DR element) of pEM-neo, generating pMSF-

neo. To shorten the outer IR/DR distance, a unique bacterial origin of

replication (RK2) and drug-resistance gene (zeocin) were employed. The

StuI – BglII fragment from the plasmid pMaRXIVfNeo, obtained from

Genetica (Cambridge, MA, USA), which contained the RK2 and zeocin-

resistance gene, was blunted and cloned into the blunted SspI – SalI

fragment of pMSF-neo, generating pMSZ-neo. The unique NotI site

within all of these transposon vectors allows for the simple replacement

of the transgene expressed by the endothelin-1 promoter. The GFP gene

from plasmid pGreenLantern, purchased from Gibco (Rockville, MD,

USA), as well as a PCR-generated SEAP gene bounded by NotI sites, were

cloned into pMSZ, generating pMSZ-GFP and pMSZ-SEAP, respectively.

The transposase expression plasmids used in these studies include

pCMV-HSB and pTRUF-HSB. Both plasmids express a humanized version

of the SB transposase (SB10) driven by either a CMV enhancer/promoter

or a CMV enhancer/chicken h-actin promoter, respectively. To generate

the humanized version of SB10 (HSB), overlapping oligonucleotides

encoding a human triplet codon-optimized version of SB10 were

annealed and ligated into plasmid pGeneOp purchased from Operon

(Alameda, CA, USA). This gene was completely sequenced to verify the

coding region. The cDNA was designed to have unique XbaI and BamHI

sites at the 5V and 3V ends, respectively. To replace SB with HSB within the

plasmid pCMV-SB (a gift from Dr. Perry Hackett, University of Minne-

sota), the XbaI –BamHI fragment of HSB was blunted and cloned into

SacII-digested and blunted pCMV-SB. Orientation of the inserted HSB was

confirmed with PstI. The CMV enhancer/chicken h-actin promoter con-

taining plasmid pTR-UF11 [33] was cut with XbaI and BamHI and the

cDNA encoding HSB was inserted directly.

Cell culture and transient transfection. HeLa (cervical), NIH3T3 (fibro-

blasts), A7R5 (smooth muscle), and transformed HUVE cells were grown

in DMEM high glucose purchased from Invitrogen (Carlsbad, CA, USA),

supplemented with 10% fetal bovine serum from JRH Bioscience (Lenexa,

KS, USA) and penicillin/streptomycin/glutamine (Invitrogen). Transient

transfection of the HeLa, NIH3T3, and A7R5 cells was performed in 6-cm

dishes using LipofectAMINE Plus (Invitrogen) reagent and 3 Ag of plasmid

DNA in serum-free medium per the manufacturer’s recommendations.

HUVECs (6-cm dishes) were transfected with GeneJammer from Strata-

gene (La Jolla, CA, USA) using 3 Ag of plasmid in serum-containing

medium.

Transposition assays. HeLa cells were transiently cotransfected with

equal molar amounts (0.46 nmol) of transposon and transposase-express-

ing plasmid or control non-transposase-expressing plasmid (pGreenLan-

tern; Gibco). If needed, additional DNA (pUC19) was added to bring the

total mass of plasmid DNA transfected to each 6-cm dish to 3 Ag. Two

days after transfection, the cells were trypsinized and counted. A total of

30,000 cells per 10-cm dish were plated in duplicate in medium con-



taining 1 mg/ml G418. Cells were incubated without disruption for 7

days, followed by medium change with fresh drug for an additional 7

days. Following 14 days of drug selection, cells were washed with cold

PBS, fixed for 15 min in 3.7% formaldehyde (in PBS), stained for 30 min

with 0.35% methylene blue (in PBS), and rinsed with water. Individual

colonies on each plate were manually counted from three independent

experiments, and the means and standard deviations from these counts

were plotted.

FACS analysis. Two days following transient transfection with either the

CMV-driven GFP plasmid pGreenLantern or the endothelin-1-driven GFP

plasmid pMSF-GFP, cells (NIH3T3, A7R5, HUVEC) were taken for FACS

analysis using Becton –Dickinson FACScan (San Jose, CA, USA). The data

were analyzed using WinMDI software plotting forward scatter versus GFP

fluorescence.

Animals and in vivo gene delivery. Five-week-old C57Bl/6 –SCID mice

purchased from Jackson Laboratories (Bar Harbor, ME, USA) were housed

under specific-pathogen-free conditions. The animals were treated

according to the NIH Guidelines for Animal Care with approval of the

IACUC of the University of Florida. Linear 22-kDa PEI (ExGen 500) from

MBI Fermentas (Hanover, MD, USA) was complexed with 60 Ag of

plasmid DNA using a charge ratio of 6 (charge ratio is expressed as PEI

nitrogen to DNA phosphate). Using a constant amount (30 Ag) of

transposon (pMSZ-SEAP) and differing amounts (0, 2, 15, and 30 Ag) of

transposase (pTRUF-HSB), the total DNA administered was maintained at

60 Ag using the plasmid pcDNA3.1 as nonspecific filler. PEI – DNA

complexes were prepared in 5% sterile glucose per the manufacturer’s

instructions. Briefly, 60 Ag of plasmid and 10.8 Al of linear PEI were

separately diluted in 100 Al of 5% glucose. The diluted PEI was then

added to the diluted DNA, vortexed, and centrifuged briefly. DNA –PEI

complexes were allowed to form at room temperature for 10 min before

injection into the mice via the tail vein (200 Al/mouse). The plasmid

DNA utilized in these studies was purified using a column chromatogra-

phy technique as previously described [34]. For the studies involving

SEAP, serial blood sampling was obtained via tail bleeds on days +1, 2, 3,

5, 7, and 14 and every 2 weeks thereafter up to day +70. For the GFP

localization studies, 40 Ag of pMSZ-GFP and 20 Ag of pTRUF-HSB were

injected and mice were evaluated for GFP expression as described below.

SEAP assay. Following tail bleeding, the samples were microcentrifuged

(�8000 rpm) at 4jC for 15 min and the serum was stored at �20jC. SEAP

activity was determined using a chemiluminescence detection kit from

Roche, Molecular Biochemicals (Mannheim, Germany) following the

manufacturer’s recommendations. Briefly, samples (10 Al) were diluted

(1:5) in 1� reaction buffer and incubated for 30 min at 65jC using a

heating block. After heat inactivation of contaminating alkaline phos-

phatases, the samples were centrifuged in a microfuge tube for 30 s at

room temperature and cooled on ice for 2–3 min. The samples were then

transferred to a 96-well plate and 50 Al of inactivation buffer was added.

After a 5-min incubation period at room temperature, 50 Al of substrate

reagent was added and the incubation was continued for an additional 10

min with gentle rocking. The plate was then read by a FLEX Station

Benchtop Scanning Fluorometer from Molecular Devices Corp. (Sunny-

vale, CA, USA) and the light signal was integrated for 1 s. The amount of

SEAP protein for each sample was quantified by comparison to a standard

curve. The mean and standard error of measurement for each experimen-

tal group was plotted utilizing Prism 4 software from GraphPad (Aurora,

CO, USA).

Immunofluorescence. Animals that received the GFP transposon were

sacrificed at day +3 or 6 weeks postinjection. Following anesthesia, the

mice were perfused with ice-cold PBS and 4% paraformaldehyde (PFA) in

PBS via cardiac puncture. The lungs were inflated with this fixative

followed by diluted Tissue Tek Optimal Cutting Temperature compound

in PBS in situ to avoid collapse. Tissues (heart, liver, spleen, lung, and

kidney) were harvested and postfixed in 4% PFA for 1 h. Following

immersion in 30% sucrose in PBS at 4jC overnight, the tissues were

quickly frozen in liquid nitrogen-cooled n-methylbutane. Cryosections (6

Am) were cut and tissues were permeabilized by incubation with 100%

methanol at �20jC for 5 min. After being blocked with 10% normal goat

serum containing PBS for 1 h, samples were incubated with primary

antibodies overnight at 4jC. The primary antibodies included a rabbit

anti-human von Willebrand factor (vWF) (1:600 dilution) from Dako

(Glostrup, Denmark) and a rabbit anti-human surfactant protein A (SP-

A) (1:3000) from Chemicon International (Temecula, CA, USA). After four

10-min washes with PBS, Alexa Texas red-conjugated anti-rabbit IgG from

Molecular Probes (Eugene, OR, USA) at 1:300 and Hoechst nuclear dye

(Molecular Probes) at 1 Ag/ml were added. Glass coverslips were mounted

using ProLong Antifade kit (Molecular Probes) and samples were imaged

with a Spot camera attached to a Nikon Eclipse 1000 microscope. Images

were printed using Adobe PhotoShop 5.0.

To quantify the percentage of GFP-expressing cells and to characterize

their cell type, multiple images (>15) of lung tissue from two independent

animals were evaluated at both the 3-day and the 6-week time points. To

calculate the percentage, the number of GFP-fluorescent cells over the

total number of cell nuclei (Hoechst) was enumerated. For the cell type

determination studies, multiple images (f12) were collected from lung

tissue stained with either the vWF or the SP-A antibody. The percentage of

cells expressing both GFP and vWF, or both GFP and SP-A, relative to total

GFP-positive cells was calculated.

ACKNOWLEDGMENTS

We thank Dr. Perry Hackett for the original Sleeping Beauty plasmids and Dr.

Sean Sullivan for the gift of the endothelin-1 promoter construct and for his

advice regarding nonviral gene delivery. James Baus provided technical

assistance. This work was supported in part by a VA Medical Research Service

award to B.S.F.

RECEIVED FOR PUBLICATION DECEMBER 24, 2003; ACCEPTED APRIL 7, 2004.

REFERENCES

1. Kay, M. A., Glorioso, J. C., and Naldini, L. (2001). Viral vectors for gene therapy: the art

of turning infectious agents into vehicles of therapeutics. Nat. Med. 7: 33 – 40.

2. Mah, C., Byrne, B. J., and Flotte, T. R. (2002). Virus-based gene delivery systems. Clin.

Pharmacokinet. 41: 901 – 911.

3. Niidome, T., and Huang, L. (2002). Gene therapy progress and prospects: nonviral

vectors. Gene Ther. 9: 1647 – 1652.

4. Ivics, Z., Hackett, P. B., Plasterk, R. H., and Izsvak, Z. (1997). Molecular reconstruction

of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells.

Cell 91: 501 – 510.

5. Izsvak, Z., Ivics, Z., and Plasterk, R. H. (2000). Sleeping Beauty, a wide host-

range transposon vector for genetic transformation in vertebrates. J. Mol. Biol. 302:

93 – 102.

6. Fischer, S. E., Wienholds, E., and Plasterk, R. H. (2001). Regulated transposition of a fish

transposon in the mouse germ line. Proc. Natl. Acad. Sci. USA 98: 6759 – 6764.

7. Dupuy, A. J., Fritz, S., and Largaespada, D. A. (2001). Transposition and gene disrup-

tion in the male germline of the mouse. Genesis 30: 82 – 88.

8. Yant, S. R., Meuse, L., Chiu, W., Ivics, Z., Izsvak, Z., and Kay, M. A. (2000). Somatic

integration and long-term transgene expression in normal and haemophilic mice using

a DNA transposon system. Nat. Genet. 25: 35 – 41.

9. Montini, E., et al. (2002). In vivo correction of murine tyrosinemia type I by DNA-

mediated transposition. Mol. Ther. 6: 759 – 769.

10. Belur, L. R., et al. (2003). Gene insertion and long-term expression in lung mediated by

the Sleeping Beauty transposon system. Mol. Ther. 8: 501 – 507.

11. Izsvak, Z., and Ivics, Z. (2004). Sleeping Beauty transposition: biology and applications

for molecular therapy. Mol. Ther. 9: 147 – 156.

12. Izsvak, Z., Khare, D., Behlke, J., Heinemann, U., Plasterk, R. H., and Ivics, Z. (2002).

Involvement of a bifunctional, paired-like DNA-binding domain and a transpositional

enhancer in Sleeping Beauty transposition. J. Biol. Chem. 24: 24.

13. Cui, Z., Geurts, A. M., Liu, G., Kaufman, C. D., and Hackett, P. B. (2002). Structure-

function analysis of the inverted terminal repeats of the Sleeping Beauty transposon.

J. Mol. Biol. 318: 1221 – 1235.

14. Geurts, A. M., et al. (2003). Gene transfer into genomes of human cells by the Sleeping

Beauty transposon system. Mol. Ther. 8: 108 – 117.

15. Zayed, H., Izsvak, Z., Walisko, O., and Ivics, Z. (2004). Development of hyper-

active Sleeping Beauty transposon vectors by mutational analysis. Mol. Ther. 9:

292 – 304.



16. Vigdal, T. J., Kaufman, C. D., Izsvak, Z., Voytas, D. F., and Ivics, Z. (2002). Common

physical properties of DNA affecting target site selection of Sleeping Beauty and other

Tc1/mariner transposable elements. J. Mol. Biol. 323: 441 – 452.

17. Cho, J., Lim, W., Jang, S., and Lee, Y. (2003). Development of an efficient endothelial

cell specific vector using promoter and 5V untranslated sequences from the human

preproendothelin-1 gene. Exp. Mol. Med. 35: 269 – 274.

18. Inoue, A., Yanagisawa, M., Takuwa, Y., Mitsui, Y., Kobayashi, M., and Masaki, T.

(1989). The human preproendothelin-1 gene: complete nucleotide sequence and reg-

ulation of expression. J. Biol. Chem. 264: 14954 – 14959.

19. Bu, X., and Quertermous, T. (1997). Identification of an endothelial cell-specific regu-

latory region in the murine endothelin-1 gene. J. Biol. Chem. 272: 32613 – 32622.

20. Goula, D., Benoist, C., Mantero, S., Merlo, G., Levi, G., and Demeneix, B. A. (1998).

Polyethylenimine-based intravenous delivery of transgenes to mouse lung. Gene Ther.

5: 1291 – 1295.

21. Zou, S. M., Erbacher, P., Remy, J. S., and Behr, J. P. (2000). Systemic linear polyethy-

lenimine (L-PEI)-mediated gene delivery in the mouse. J. Gene Med. 2: 128 – 134.

22. Rudolph, C., Lausier, J., Naundorf, S., Muller, R. H., and Rosenecker, J. (2000). In vivo

gene delivery to the lung using polyethylenimine and fractured polyamidoamine den-

drimers. J. Gene Med. 2: 269 – 278.

23. Oh, Y. K., Kim, J. P., Yoon, H., Kim, J. M., Yang, J. S., and Kim, C. K. (2001). Prolonged

organ retention and safety of plasmid DNA administered in polyethylenimine com-

plexes. Gene Ther. 8: 1587 – 1592.

24. Berger, J., Hauber, J., Hauber, R., Geiger, R., and Cullen, B. R. (1988). Secreted pla-

cental alkaline phosphatase: a powerful new quantitative indicator of gene expression

in eukaryotic cells. Gene 66: 1 – 10.

25. Cullen, B. R., and Malim, M. H. (1992). Secreted placental alkaline phosphatase as a

eukaryotic reporter gene. Methods Enzymol. 216: 362 – 368.

26. Thyagarajan, B., Olivares, E. C., Hollis, R. P., Ginsburg, D. S., and Calos, M. P. (2001).

Site-specific genomic integration in mammalian cells mediated by phage phiC31 in-

tegrase. Mol. Cell. Biol. 21: 3926 – 3934.

27. Goula, D., et al. (2000). Rapid crossing of the pulmonary endothelial barrier by poly-

ethylenimine/DNA complexes. Gene Ther. 7: 499 – 504.

28. Ma, Z., et al. (2002). Lipid-mediated delivery of oligonucleotide to pulmonary endo-

thelium. Am. J. Respir. Cell Mol. Biol. 27: 151 – 159.

29. Li, S., Tan, Y., Viroonchatapan, E., Pitt, B. R., and Huang, L. (2000). Targeted gene

delivery to pulmonary endothelium by anti-PECAM antibody. Am. J. Physiol. Lung Cell

Mol. Physiol. 278: L504 – L511.

30. Tan, P. H., et al. (2003). Antibody targeted gene transfer to endothelium. J. Gene Med.

5: 311 – 323.

31. Hood, J. D., et al. (2002). Tumor regression by targeted gene delivery to the neo-

vasculature. Science 296: 2404 – 2407.

32. Davidson, A. E., et al. (2003). Efficient gene delivery and gene expression in zebrafish

using the Sleeping Beauty transposon. Dev. Biol. 263: 191 – 202.

33. Xu, L., et al. (2001). CMV-beta-actin promoter directs higher expression from an ad-

eno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1

alpha promoter and results in therapeutic levels of human factor X in mice. Hum. Gene

Ther. 12: 563 – 573.

34. Hines, R. N., O’Connor, K. C., Vella, G., and Warren, W. (1992). Large-scale purification

of plasmid DNA by anion-exchange high-performance liquid chromatography. Biotech-

niques 12: 430 – 434.



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Endothelial targeting of the Sleeping Beauty transposon within lung