www.nature.com/naturebiotechnology • OCTOBER 2002 • VOLUME 20 • nature biotechnology

Transposition from a gutless adeno-transposonvector stabilizes transgene expression in vivoStephen R.Yant, Anja Ehrhardt, Jacob Giehm Mikkelsen, Leonard Meuse, Thao Pham, and Mark A. Kay*

Published online: 16 September 2002, doi:10.1038/nbt738

A major limitation of adenovirus-mediated gene therapy for inherited diseases is the instability of transgeneexpression in vivo, which originates at least in part from the loss of the linear, extrachromosomal vectorgenomes. Herein we describe the production of a gene-deleted adenovirus–transposon vector that stablymaintains virus-encoded transgenes in vivo through integration into host cell chromosomes. This system uti-lizes a donor transposon vector that undergoes Flp-mediated recombination and excision of its therapeuticpayload in the presence of the Flp and Sleeping Beauty recombinases. Systemic in vivo delivery of this sys-tem resulted in efficient generation of transposon circles and stable transposase-mediated integration inmouse liver. Somatic integration was sufficient to maintain therapeutic levels of human coagulation Factor IXfor more than six months in mice undergoing extensive liver proliferation. These vectors combine the versatili-ty of adenoviral vectors with the integration capabilities of a eukaryotic DNA transposon and should prove use-ful in the treatment of genetic diseases.

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Successful gene therapy for genetic diseases will require two majorattributes from a single vector: high-efficiency gene delivery and life-long therapeutic gene expression. Among the vector technologiescurrently available, recombinant adenovirus (Ad) vectors remainattractive vehicles for therapeutic gene transfer. These vectors can begrown to high titers, exhibit a broad tropism, can transduce dividingand nondividing cells, and are one of the most efficient vehicles forin vivo gene delivery1. However, early versions of Ad vectors wereplagued by toxicity and immunogenicity due to in vivo synthesis ofviral antigens from genes still contained in the vector2–4.

To overcome these problems, the Cre-loxP helper-dependent(HD) system was developed to generate recombinant adenovirusesin which all viral coding sequences have been deleted5. Because theHD vector requires only the inverted terminal repeats (ITRs) andpackaging signal for proper DNA replication and virus assembly,these vectors can accommodate up to 35 kilobases of foreign DNA.Importantly, these gene-deleted vectors exhibit little to no toxicity in vivo and can produce therapeutic amounts of various proteins inanimals6–12. Nonetheless, transgene expression from these non-replicative HD vectors still remains unstable in vivo, declining by asmuch as 95% over a period of one year11. Recent in vitro studies indi-cate that the frequency of genomic integration of Ad vectors is quitelow13,14, suggesting that transgene instability originates at least inpart from the loss of the linear, extrachromosomal HD genome intransduced cells.

One approach to circumvent this obstacle is to incorporate a DNAtransposon into the Ad vector to maintain Ad-encoded transgenes individing cells through genomic integration. Although integrationitself does not always guarantee long-term gene expression, it is themost reliable way to maintain genetic material within a cell overtime. Currently, the only DNA elements reported to function withinthe context of mammalian cells are members of the Tc1/marinerfamily of transposable elements15. Recent studies indicate that the

most active of these members is the Tc1-like element SleepingBeauty, which was reconstructed from pieces of an ancient fish ele-ment16,17. This transposon undergoes cut-and-paste transpositionthrough a DNA intermediate, a process that requires the binding ofthe Sleeping Beauty transposase (SB) to short direct-repeat sequencesembedded in the terminal inverted repeats (IRs) of the element17.Transposition is catalyzed entirely by the SB transposase and alwaysoccurs into a TA target dinucleotide, which is duplicated upon inser-tion by cellular DNA repair pathways. Previously, we reported thatSB transposase expression allows plasmid-based transposons to inte-grate safely and stably into mouse liver DNA18. Importantly, transpo-sition was sufficient to maintain long-term transgene expression in vivo, even during periods of extensive liver regeneration19. Despiteits enormous potential, the inability to deliver this integrating systemin vivo efficiently in a clinically relevant manner remains a formida-ble obstacle to its practical implementation in basic research andhuman gene therapy applications.

In this report, we have incorporated the SB integration machineryinto Ad vectors to combine the major advantages of each system. Inthe process, we have identified a new limitation in the SB system,namely that to transpose, SB transposons need to circularize.Consequently, we have generated gene-deleted Ad vectors thatrelease circular transposons from the linear Ad genome through theactivities of the Flp/FRT recombination system. These high-capacityadeno-transposon vectors were characterized in mice and couldfacilitate the somatic integration of virus-encoded transgenes intohost cell chromosomes, resulting in greatly improved longevity ofAd-based gene expression in vivo.

ResultsAnalysis of Ad-based transposition in vitro and in vivo. To determinewhether the Sleeping Beauty transposon system could function in thecontext of an adenovirus, we studied Ad-based transposition in vivo

Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA 94305-5208.*Corresponding author (markay@stanford.edu).

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using an experimental strategy previously validated with plasmid-based vectors18. To do this, we injected C57Bl/6 mice with an E1/E3-deficient adenovirus encoding a human α1-antitrypsin transposon(Ad-ThAAT) together with a second adenovirus encoding eithertransposase (Ad-SB) or no transgene (Ad-null) as a control (Fig. 1A).We then monitored serum hAAT levels over time as a general measureof transposition in these mice. Results of these studies are shown inFigure 1B and illustrate a lack of any significant improvement in thelong-term persistence of transposon expression in Ad-SB-infectedmice. Because in vitro studies using an adenovirus encoding aneomycin transposon also did not indicate transposition (data notshown), SB transposition from conventional first-generation Ad vec-tors appears to be very inefficient, if it occurs at all.

Uncontrolled transposase production and transposon lineariza-tion both significantly impair transposition frequencies. Whileinvestigating possible cis- and/or trans-acting factor(s) that mightinhibit transposition from Ad vectors, we learned that SB overex-pression significantly impairs plasmid-based transposition in vitroand in vivo (unpublished observations). Although the mechanism

behind this inhibitory pathway remains under investigation, theseresults suggest that the absence of Ad-based transposition might bedue, at least in part, to overproduction of the transposase enzyme intransduced cells. Alternatively, it is possible that the linear viralgenomes are in some way resistant to efficient SB transposition, evenin the absence of any associated proteins. For instance, in vitro stud-ies with the Tc1 transposon have shown that the frequency of trans-

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Figure 1. Analysis of transposition from E1/E3-deleted adenoviruses andlinear transposon DNA. (A) Structures of the E1/E3-deleted transpositionvectors Ad-SB and Ad-ThAAT for in vivo delivery of the Sleeping Beautytransposase–transposon system. 5’ITR and 3’ITR, Left and right terminiof adenovirus type 5, respectively; RSV, Rous sarcoma virus long terminalrepeat promoter; SB, Sleeping Beauty transposase; pA, polyadenylationsignal; E1/E3, adenovirus type 5 early regions 1 and 3, respectively; IR,Sleeping Beauty inverted repeat sequences; hAAT, human α1-antitrypsincDNA. (B) Long-term Ad-based transposon expression in mice in thepresence and absence of transposase. C57Bl/6 mice (n = 5 mice pergroup) were injected through the tail vein with 2 × 109 transducing units(TU) AdThAAT together with 6 × 109 TU of either AdSB (�) encoding theSB transposase or Ad-null (�) as a control. (C) Transposition efficiencyfrom circular and linear transposable elements in cultured mammaliancells. Cells were transfected with transposon DNA and, when applicable,a helper plasmid encoding either wild-type (SB) transposase or acatalytically inactive mutant (mSB) transposase as a control. Transfectedcells were growth-selected in G418 for 14 days, fixed, stained, andcounted. The number of G418-resistant (G418r) colonies (mean ± s.d.)obtained after three independent transfections is shown. SV40, Simianvirus promoter; neo, neomycin-phosphotransferase gene. Left panel,transposition of a supercoiled neo-marked transposon in HeLa cells;middle panel, transposition frequency of a linearized neo-marked elementin HeLa cells; right panel, transposition of a linear neo-marked transposonwith (+IR) and without (–IR) flanking inverted repeats in HeLa cellsconstitutively expressing the SB transposase (HeLa-SB)49.

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Figure 2. The adeno-transposon system. (A) Overview of the strategy tofacilitate transposition from a helper-dependent adenovirus vector. An Advector containing a Sleeping Beauty transposon remainsextrachromosomal in SB-expressing cells because the transposase cannotefficiently act upon linear DNA structures. When the transposon is flankedby a pair of Flp recognition target (FRT) sequences, the Ad vectorundergoes conditional rearrangement in cells co-expressing the Flprecombinase.This results in excision of the transposon from the Adgenome and its circularization by Flp-mediated recombination. In contrastto their linear counterparts, these circular elements actively undergo DNA-mediated transposition, resulting in stable insertion of the transposon intohost cell chromosomes. Prom, Mammalian promoter. (B) Structure ofhelper-dependent transposition vectors. We used a two-vector approach toanalyze transposition from gutless Ad vectors in vivo. This strategyemploys the use of one vector to provide the Flp and SB recombinasesthat act upon a second Ad vector containing an SB donor transposon.HD-SB-Flp and HD-mSB-Flp both encode the enhanced Flp recombinase(Flpe) necessary for conditional vector rearrangement, but HD-mSB-Flpcannot support transposition because of an inactivating mutationintroduced into the transposase gene.The donor vectors HD-FRT-Tnori,HD-Tnori, HD-FRT-TLacZ, HD-TLacZ, and HD-FRT-ThFIX contain donortransposons encoding kanamycin (3.4 kb), β-galactosidase (5.5 kb), orhuman Factor IX (4.5 kb), respectively.The intron-containing transgenespresent in the LacZ and hFIX constructs are initially split and thus remaininactive until Flp-mediated circularization restores the correct readingframe. HD-Tnori and HD-TLacZ are control vectors that lack flanking FRTsites and thus cannot undergo the Flp-mediated recombination necessaryfor both circle formation and SB-mediated transposition. IgκMAR, twocopies of the immunoglobulin κ matrix attachment region; stuffer DNA, a16.2 kb fragment of alphoid repeat DNA from human chromosome 17;MTH, mouse metallothionein I gene promoter; EF1α, human elongationfactor 1α gene enhancer-promoter; Tn5, bacterial promoter; kan,kanamycin resistance gene; ori, p15A bacterial origin of replication; cam,chloramphenicol resistance gene; LacZ, recombinant split β-galactosidasegene containing intron 1 of the human chorionic gonadotropin (hCG) 6 gene inserted between nucleotides 1,761 and 1,762 of the LacZ codingsequence; ApoE/HCR, 1.2 kb fragment containing the hepatocyte controlregion (HCR) from the apolipoprotein E (ApoE) gene; hAATp, human α1-antitrypsin gene promoter; hFIX, split human Factor IX (hFIX) cDNAcontaining 1.4 kb truncated intron A from the hFIX gene.

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position from a supercoiled, circular donor transposon is reduced20-fold upon linearization20. Indeed, when we studied transpositionof circular and linear neo-marked elements in cultured mammaliancells, we found that circular SB transposons underwent transposi-tion much more readily than their linearized counterparts (Fig. 1C).Therefore, our findings collectively indicate that the production of afunctional adeno-transposon vector may require the use of regulato-ry components and additional recombination systems to facilitatethe formation of circular intermediates in the cell.

Experimental strategy to facilitate Ad-based transposition.Herein we devised a adeno-transposon system (Fig. 2A) that webelieved might overcome some of the limitations identified in theprevious section. This approach utilizes gene-deleted Ad vectorscontaining Flp recognition target (FRT) site sequences flankingthe Ad-encoded transposon. Recent reports demonstrating itsactivity in the context of both adenoviruses21 and mammals22

indicate that expression of the thermostably enhanced Flp recom-binase should result in excision of the FRT-flanked transposonDNA from the Ad genome. Importantly, DNA excision throughthe Flp/FRT recombination system also results in DNA circular-ization, and thus should provide an improved substrate for SBtransposition in transduced cells.

To investigate the potential utility of this system for stable in vivogene transfer, we constructed Ad vectors encoding either SB trans-posons or the necessary recombinases (Fig. 2B). In all cases, we pack-aged these constructs into helper-dependent vectors to avoid theproblems associated with leaky expression of viral proteins.Furthermore, transposon vectors were produced with and withoutFRT sites flanking the transposon cassette to investigate in vivo the

requirement for the formation of a circular intermediate through theFlp/FRT recombination system. We also produced two gene-deletedAd vectors that constitutively express the enhanced Flp recombinaseand inducibly express either wild-type transposase (HD-SB-Flp) orcatalytically inactive mutant SB (HD-mSB-Flp) in the presence ofzinc. Based on their potential ability to control transposase produc-tion and to release circular transposon molecules, these low-immunogenic vectors are significantly more suitable for SB transpo-sition than their first-generation predecessors.

Recovery and sequence analysis of vector integration sites. Todetermine whether Sleeping Beauty could function in the context ofthese adeno-transposon vectors, we administered HD-Tnori andHD-FRT-Tnori to mouse livers and induced transposition followingco-injection with HD-SB-Flp or HD-mSB-Flp as a control. We thenidentified transposition events using a plasmid recovery strategy thatfacilitates the cloning and sequencing of cellular–transposon junc-tion fragments (Fig. 3A). Interestingly, when we analyzed total liverDNA from mice injected with HD-FRT-Tnori and HD-SB-Flp(group 4), we obtained bacteria that were predominantly cams andkanr (62% or 21/34), suggesting efficient transposon excision fromthe camr donor (Fig. 3B). Plasmid DNA analysis from eight of thesecams/kanr bacteria showed that each clone contained two bands (2.4and 0.4 kilobases) corresponding to internal transposon sequences,as well as a variety of novel bands indicative of transposase-mediatedintegration (Fig. 3C). Sequence analyses showed that each end of thetransposon was flanked by TA dinucleotides, followed by sequencesthat were different from the Ad vector sequences originally flankingthe transposon in HD-FRT-Tnori (Fig. 3D). These findings are con-sistent with a cut-and-paste mode of transposon integration23. Whenwe screened the sequences flanking the transposon in these cams

clones for homologies in both GenBank and the Ensembl mousegenome database by a BLAST search, we found that integrationoccurred into multiple different chromosomes. Cellular sequencescontained in one clone possessed 98% homology to intron 2 of themouse mSP100-rs1 gene on chromosome 1 (clone 2; positivity,496/505; GenBank accession no. AF176311), whereas another cloneexactly matched intron 3 of the mouse Mypt1 gene on chromosome10 (clone 7; positivity, 906/906; ENSMUSG no. 19907). All otherclones showed significant homologies to either mouse repetitive ele-ments or to currently undefined mouse sequences. Because chromo-somal deletions and rearrangements have been observed at the site ofrecombinant adeno-associated virus (AAV) provirus integration24,25,we carefully analyzed the genomic sequences at the point of transpo-son insertion but could find no alteration of these sites other thanthe duplication of the TA target dinucleotide.

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Figure 3. Conditional rearrangement and chromosomal integration of HDvectors in vivo following expression of the Flp and SB recombinases.(A) Overview of the genetic approach used to recover transposons frommouse chromosomes. C57Bl/6-scid mice (n = 2 mice per group) wereinjected into the tail vein with 1 × 109 TU of each virus. We inducedtransposase expression in a stepwise manner during the five weeksimmediately following vector administration by addition of increasingconcentrations of ZnSO4 to the animal drinking water. Groups 1–3 wereincluded as controls to demonstrate that transposon integration from an Advector requires both circle formation and transposase activity. (B) Structureof the predicted excised circular episome. Numbers represent HindIIIfragment sizes in kilobases. H, HindIII. (C) Transposon DNA analysis byHindIII digestion and ethidium bromide gel electrophoresis. Lanes 1–8, DNAfrom eight independent kanr/cams clones recovered from the liver DNA ofgroup 4 (HD-FRT-Tnori + HD-SB-Flp) mice. Arrows indicate internaltransposon-specific fragments (2.4 kb and 0.4 kb). Size markers are inkilobases. (D) Transposon insertion site sequences. Target site duplicationsare shown in bold uppercase, novel flanking sequences are in lowercase,and transposon sequences are denoted by the central shaded box. Theorigin and identity of transposon flanking sequences, shown to the right,were identified through mouse genome database homology searches.

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In contrast to the injection of HD-FRT-Tnori and HD-SB-Flp ade-novirus vectors, liver DNA from control mice (groups 1–3) producedvery few cams/kanr bacterial colonies, and DNA from each of theseclones did not contain any transposon-specific fragments. Theabsence of detectable transgene integration in the absence of either anexcised transposon circle (groups 1–2) or transposase activity (groups2–3) indicates that transposition from an adenovirus requires thecoordinated activities of the Flp and SB recombinases. Collectively,these data demonstrate that Sleeping Beauty can stably integrate Ad-encoded transgenes into the genome of mouse hepatocytes in vivo.

Flp-Dependent reporter gene activation and persistence invivo. We studied the activity of the adeno-transposon system inmouse hepatocytes by analyzing the persistence of expressionfrom a β-galactosidase transposon in the presence and absence ofthe Flp and SB recombinases. Importantly, we have physically separated the 5′ and 3′ portions of the LacZ gene in the parentalvectors to ensure that proper mRNA splicing and β-galactosidaseexpression only occurs when a circular DNA molecule is formed inthe cell (see Fig. 2B). We injected immune-deficient mice with anAd vector encoding an inactive LacZ transposon with (HD-FRT-TLacZ) or without (HD-TLacZ) flanking FRT sites together witheither HD-SB-Flp or HD-mSB-Flp as a control. We then inducedtransposase expression during the next five weeks and analyzedthe liver for β-galactosidase expression after its isolation during asurgical two-thirds partial hepatectomy (PH). Under these experi-mental conditions, ∼ 45% of mouse hepatocytes were found tocontain transposon circles at this time point (Fig. 4C, D), com-pared with just 1–2% in control mice that either did not receive anFRT-containing reporter gene (Fig. 4A) or did not receive a Flprecombinase gene (Fig. 4B). Therefore, transposase and transpo-son circles can be efficiently co-delivered in vivo using a two-component Ad-based system.

To facilitate the loss of nonintegrated trans-poson forms in the liver, we induced additionalhepatocellular cell cycling after the PH byrepeatedly injecting these animals with carbontetrachloride (CCl4) over the next 13 weeks andthen analyzed liver sections for β-galactosidaseexpression. Results of these studies showed thepresence of rare isolated X-gal+ hepatocytes ineach of the control groups (Fig. 4E–G), and amaximum of three X-gal+ hepatocytes within asingle cluster. Interestingly, cross-sectionalanalysis of liver tissue from mice that receivedHD-FRT-TLacZ and HD-SB-Flp showed thepresence of many large X-gal+ clusters, eachconsisting of ∼ 10–70 hepatocytes (Fig. 4H, I),the formation of which is consistent with anintegration event occurring sometime beforethe induction of hepatocellular regeneration.Moreover, even though the number of thesefoci cannot be used to determine the exact inte-gration frequency in vivo because CCl4 inducesa nonuniform proliferative response, we havefound that SB-expressing mice formed ∼ 11-fold more blue foci (arbitrarily defined as≥3 clustered blue hepatocytes) compared withmice expressing the mutant transposase.Therefore, these data collectively demonstratethat transposase-mediated integration canmaintain the expression of Ad-encoded trans-genes during cell cycling in vivo.

Genomic integration stabilizes expressionof a Factor IX transposon in mice undergo-

ing hepatic cell cycling. To explore its potential as a therapeuticagent, we tested whether integration using this adeno-transposonsystem could maintain long-term production of human coagula-tion Factor IX (hFIX) in adult mice. To do this, we injected HD-FRT-ThFIX into C57Bl/6 mice together with either HD-SB-Flp orHD-mSB-Flp as a control, and then monitored the production ofhuman Factor IX after inducing transposase expression for threeweeks. Based on the structural design of the HD-FRT-ThFIX vec-tor (see Fig. 2B), Factor IX expression will only originate from cir-cular molecules that are excised from the Ad genome by the Flprecombinase. To facilitate the loss of nonintegrated transposonforms, we repeatedly induced hepatocellular regeneration in thesemice by first doing a surgical two-thirds partial hepatectomy andthen by injecting CCl4. Approximately six months after vectoradministration, human FIX levels were nearly undetectable (1–2ng/ml) in control mice injected with HD-mSB-Flp (n = 5), where-as mice receiving HD-SB-Flp still had stable human FIX levelsranging from 83 ng/ml to 186 ng/ml (n = 5) (Fig. 5). This corre-sponds to ∼ 135-fold more Factor IX in the long term in SB-expressing mice compared with control mice receiving nontrans-posable vectors and is within a range that has been previouslyshown to be therapeutic in a mouse model of hemophilia B18.Collectively, these results suggest that the frequency of chromoso-mal integration through the adeno-transposon system is sufficientto provide significantly improved transgene longevity from gene-deleted Ad vectors.

Toward the development of a single integrating adeno-transpo-son vector. To investigate whether this integrative system could besafely packaged into a single high-capacity adenovirus, we producedthe Ad vector HD-FRT-TNC-SB, which encodes a neo-markedtransposon and a promoterless transposase gene that becomes acti-vated upon circle formation (Fig. 6A). This strategy prevents trans-

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Figure 4. Adenovirus-based β-galactosidase expression in mouse liver before and afterhepatocellular regeneration. C57Bl/6-scid mice were injected into the tail vein with 1 × 109 TU eachof HD-TLacZ and HD-SB-Flp (A, E), HD-FRT-TLacZ alone (B, F), HD-FRT-TLacZ and HD-mSB-Flp(C, G), or HD-FRT-TLacZ and HD-SB-Flp (D, H). Transposase expression was induced in a stepwisemanner during the five weeks immediately following vector administration by addition of increasingconcentrations of ZnSO4 to the animal drinking water. Representative sections are shown from liverremoved during a surgical two-thirds partial hepatectomy (PH) 5 weeks postinjection (p.i.) (A–D;n = 5 mice per group) and again after an additional 16 weeks (E–I; n = 3 mice per group). All animalsreceived intraperitoneal injections of CCl4 at 8, 10, 12, 15, 17, and 19 weeks after vectoradministration to further promote hepatocellular cell cycling. Arrows denote the presence of β-galactosidase+ foci, examples of which are shown in more detail in the six lower (I) panels.Magnifications: A–D, original ×100; E–H, original ×40; I, originals ×200. Bars, 500uM. Bar in Dapplies to A-D and bar in H applies to E-H.

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posase overproduction from the parental vector and should ensurethe elimination of transposase sequences over time. Althoughmethods to control Flp activity during vector production remain indevelopment, we expressed Flp protein in trans to study the poten-tial of this transposase activation approach. To do this, we injectedmice with HD-FRT-TNC-SB and tested for transposon integrationby a plasmid recovery strategy two months after systemic delivery ofa plasmid encoding the Flp recombinase. Although there wereinstances in which the transposon jumped into the Ad vector itself(n = 3/10), the majority of the insertions (70%) were found to bewithin mouse chromosomal DNA (Fig. 6B). These results demon-strate the feasibility of using recombinase-activated transposaseexpression to achieve stable integration in vivo from a single high-capacity Ad vector.

DiscussionHere we have identified circular transposon forms as the optimalsubstrate for SB-mediated transposition and have developed effi-cient ways to deliver these circles in vivo by combining a site-specificrecombination system with a gene-deleted adenovirus. Although it isstill unclear how transposon circularization promotes transposition,studies in other transposon systems suggest that supercoiling ofclosed circular donor DNA may promote structural features in theDNA that favor either interactions with proteins and/or the orderedassembly and activation of the transposase–transposon complex26–28.Importantly, we have shown that this adeno-transposon systemcould integrate transgenes into the liver chromosomes of adult mice,likely resulting in lifelong gene maintenance in vivo. These findingsdemonstrate DNA-mediated transposition from an adenovirus andprovide an integrative strategy for high-efficiency gene transfer andstable transduction in vivo.

This vector system offers a number of important advantages overexisting chimeric vector systems previously developed to maintainadenovirus transgenes through integration into host cell chromo-somes29–34. First, in marked contrast to Ad-based vectors that relyon the AAV integration machinery for stable transduction30,31,35,transposase-mediated integration does not induce chromosomaldeletions and/or rearrangements at the site of transgene integra-tion24,25. Second, unlike the Ad–retrovirus33,34,36 and Ad–retrotrans-

poson29 vector systems, our integrative transposon system does notrequire a reverse-transcription step that can produce truncatedforms of the transgene. Furthermore, both of these Ad-retro sys-tems require a two-stage infection process in vivo to stably integratethe transgene in neighboring, actively replicating cells.Importantly, although our system requires an additional circular-ization step, we have found that cell cycling is not required for SB-mediated transposition in the liver (unpublished), indicating thatthe adeno-transposon system can function directly within thepostmitotic tissues that it infects. Finally, despite all these differentintegrative systems that have been developed, the only reported invivo studies thus far have been with an E1-deleted Ad vector encod-ing a luciferase gene flanked by retroviral elements32. Surprisingly,this vector (AdLTR) was found to integrate in rat brain tissue in theabsence of exogenous integrase. This suggests that the integrativeprocess used by this vector may involve recombination withendogenous retroviral sequences, which could result in the inser-tion of neighboring viral sequences as well. In contrast, we utilizeda low-immunogenic gene-deleted vector and have identified SBtransposition as the mechanism of transgene persistence in mouseliver. In addition, we also followed the long-term production (morethan six months) of a clinically relevant reporter gene (Factor IX)in an immune-competent host and did so under conditions ofextensive hepatocellular regeneration so as to test stringently itsability to maintain transgenes over time. Based on these encourag-ing results, we have begun developing new adeno-transposon vec-tors for stable transgene insertion using a single gene-deleted vec-tor. With further development, it might eventually be possible tocontrol the level of DNA integration by regulating production ofboth the Flp and SB recombinases.

Although recent studies indicate that high-level SB expression isnot cytotoxic (unpublished), we have taken precautions to ensurecontrolled transposase production from our integrative vector sys-tem. Furthermore, despite high transposition rates (∼ 20%) in thespermatids of transgenic mice16, the actual frequency in non-germ-cell tissues may be significantly lower (≤10–6) based on quan-titative estimates in both embryonic stem (ES) cells37 and culturedhuman cells (unpublished). Importantly, the theoretical risk ofgerm-line transmission using our system may be quite small consid-ering that spermatids are highly refractory to infection by aden-ovirus38,39. Moreover, with the current vector design, expression ofthe transposase is transient because of degradation or loss of thetransposase gene during cell division. With that said, application ofthis integrative system to transgenic mice ubiquitously expressingthe Coxsackie adenovirus receptor40 might actually provide newmeans to produce stable transgenics.

Based on the 29 transposition events we have isolated thus far,in vivo integration from the SB system appears to be random.Therefore, it might be useful in future studies to investigate whateffect, if any, insulators41 have on long-term transposon expression,considering that a subset of the total events probably occur at tran-scriptionally inactive loci. Alternatively, it might be useful to employalternative recombination systems in this system, such as the φC31phage recombinase that mediates site-specific integration in cul-tured mammalian cells42,43. Although genomic integration representsan important step toward lifelong gene expression using Ad vectors,ongoing research should help determine what role, if any, immuneresponses to the Ad capsid and/or the encoded transgenes have onlong-term transgene persistence in vivo.

In summary, our data indicate that integration through theadeno-transposon system can considerably reduce the need for vec-tor readministration. With their ability to transduce dividing andnondividing cells efficiently and stably, these gene-deleted vectorsprovide a potential new means to treat genetic diseases.

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Figure 5. In vivo human Factor IX persistence in actively dividing mouselivers via the adeno-transposon system. C57Bl/6 mice (n = 5 mice pergroup) were injected into the tail vein with 5 × 108 TU of HD-FRT-ThFIXtogether with 5 × 108 TU of either HD-SB-Flp (�) or HD-mSB-Flp (�) as acontrol. Transposase expression was induced in treated animals duringthe first three weeks immediately following vector administration byaddition of drinking water containing ZnSO4 (25 mM final). Approximatelythree weeks postinjection, the zinc was removed from the water and asurgical two-thirds partial hepatectomy (PH) was done to stimulatehepatocellular regeneration and loss of episomal vectors. Arrows indicatewhen animals received intraperitoneal injections of CCl4 to furtherpromote hepatic cell cycling.

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Experimental protocolAnimal studies. We obtained six-week-old C57Bl/6 and C57Bl/6-scid micefrom Jackson Laboratory (Bar Harbor, ME). Animals were treated accordingto the NIH Guidelines for Animal Care and the guidelines of StanfordUniversity. Mice were anesthetized with isoflurane (Abbott Laboratories,Abbott Park, IL) for surgical partial hepatectomy, which uniformly inducesone to two rounds of hepatic cell division throughout the liver over a period of three weeks. We diluted CCl4 in mineral oil and injected itintraperitoneally into the abdomen of mice. This drug kills the cells in whichit is metabolized, resulting in a compensatory proliferative response in thesurrounding areas of the liver44. For transposase induction, we added ZnSO4

(25 mM final) to the animal drinking water following vector administra-tions41. Adenoviruses were diluted in PBS and injected into the tail vein.Plasmid DNAs were delivered to mice by hydrodynamics-based transfection

as described45,46. We collected mouse serum from the retro-orbital plexusand analyzed it by an ELISA for total hAAT or hFIX antigens as described18.

Generation of E1/E3-deleted adenoviruses. Details of the construction ofthe adenoviral shuttle plasmids used for production of AdSB, AdThAAT(encodes a 2.8 kb hAAT transposon), and AdTnori (encodes a 3.4 kbneomycin-marked transposon) are available upon request. The E1/E3-deleted Ad vectors AdSB, AdThAAT, and AdTnori were prepared followingco-transfection with pJM17 as described47. We amplified viral plaques in293 cells, analyzed adenoviral DNA by HindIII digestion, and purifiedviruses to high titer by two-step CsCl ultracentrifugation48. These viruseswere titered by optical density (OD) measuring according to the estimationthat one OD260 = 1 × 1012 transduction units (TU)/ml.

Production of high-capacity Ad vectors. The contents of each Ad vector aredescribed in Figure 2. Details of the construction of all helper-dependent(HD) adenovirus vectors are available upon request. HD adenovirus vectorswere produced as described11. We used a plaque-forming assay to determinethe level of helper-virus contamination in our viral preps (typically ≤0.2%)and titered each HD vector by quantitative Southern blot analysis using viralDNA isolated from virally infected HeLa cells.

Cell culture studies. To analyze transposition from supercoiled and linearsubstrates, we transfected 5 × 105 HeLa cells with 1.5 µg of pCMV-SB orpCMV-mSB18 together with either 1.5 µg pTnori-Cam or 1.5 µg of a gel-purified NdeI/AccI Tnori fragment using Superfect (Qiagen, Valencia, CA). Ina follow-up study, we transfected 5 × 105 HeLa-SB cells49 with 3 µg of eitherNdeI/XbaI-digested pTnori (retains both IRs) or NdeI/BamHI-digestedpTnori (removes the 3′ IR/DR structure). In all cases, we trypsinized cells 48 h later and then growth-selected in DMEM containing G418 (500 µg/ml)for 14 days. We counted the number of G418r colonies on each plate after fix-ing in formaldehyde and staining with methylene blue.

Transposon recovery from the mouse genome. We injected C57Bl/6-scid micewith 1 × 109 TU each of HD-Tnori and HD-SB-Flp, HD-FRT-Tnori alone,HD-FRT-Tnori and HD-mSB-Flp, HD-FRT-Tnori and HD-SB-Flp, inducedtransposase expression for five weeks by addition of increasing concentrationsof ZnSO4 to the animal drinking water, and then killed animals for liver DNAisolation. For the single integrating vector, we injected C57Bl/6-scid mice firstwith 2 × 109 TU of HD-FRT-TNC-SB and then with 25 µg pOG-Flpe22 oneweek later to stimulate vector rearrangement and transposase expression.These mice were killed seven weeks later for DNA analysis. In all cases, we useda plasmid rescue strategy previously described18 to isolate flanking genomicsequences from mouse chromosomes, and screened them against the mousegenome database (www.ensembl.org/Mus_musculus/) to identify homolo-gous mouse sequences.

β-galactosidase expression. Mouse liver tissue was frozen in OCT buffer ondry ice. We stained liver sections (10 µm) for β-galactosidase expression using5-bromo-4-chloro-3-indolyl β-D-galactoside (X-gal) and counted X-gal+-hepatic nuclei from two to three liver lobes using a light microscope.

AcknowledgmentsWe thank K. Ohashi for helpful discussions, J. Chamberlain for providing theC7-Cre cells, and S. Dymecki for providing Flp/FRT-based plasmids. This workwas supported by NIH grant DK49022 (M.A.K.). A.E. is the recipient of aJudith Pool National Hemophilia Fellowship. J.G.M. was funded by grants fromthe Carlsberg Foundation and the Danish Medical Research Council.

Competing interests statementThe authors declare that they have no competing financial interests.

Received 20 May 2002; accepted 16 July 2002

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nature biotechnology • VOLUME 20 • OCTOBER 2002 • www.nature.com/naturebiotechnology1004

Figure 6. Transposition in vivo from a single Ad vector encodingtransposon and Flp-activated transposase activities. (A) Experimentalstrategy for controlled transposase expression from a single Ad-basedtransposition vector. A linear adenoviral vector (HD-FRT-TNC-SB)containing the entire Sleeping Beauty transposon system flanked by apair of FRT sites forms a circular intermediate in the presence of theFlp recombinase. Circularization of the transposition cassette results incis-activation of transposase expression and genomic transpositionfrom the adenoviral circle. In this configuration, the episome-encodedtransposase gene is either degraded after transposition or lost overtime during cell division. CMV, Minimal cytomegalovirus core promoter.(B) Transposon insertion site sequences from mice injected with asingle integrating adenovirus vector. C57Bl/6-scid mice (n = 3) wereinjected with 2.4 × 109 TU of HD-FRT-TNC-SB through the tail vein.One week later, animals were injected with 25 µg pCMV-Flpe topromote Flp-mediated vector rearrangement, and seven weeks later,total liver DNA was isolated and used to recover integratedtransposons through a plasmid recovery strategy. Target siteduplications are shown in bold uppercase, novel flanking sequencesare in lowercase, and transposon sequences are denoted by thecentral shaded box.

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