NEWS AND VIEWS

www.nature.com/naturebiotechnology • OCTOBER 2002 • VOLUME 20 • nature biotechnology 987

Recombinant adenoviruses have beenamong the most widely explored vectorsystems for delivering genes to mammaliancells. A particular difficulty in regard togene-deleted adenoviral vectors has beenthe lack of persistence of transgene expres-sion in vivo—specifically in organs (such asliver) that can undergo substantial regener-ation—resulting in the gradual loss ofextrachromosomal vector DNA. In thisissue, Kay and colleagues1 report a systemthat addresses the challenge of genomepersistence after adenovirus-mediated,

liver-directed gene transfer. Their report isan extension of earlier work by the samegroup demonstrating the potential utilityof a member (Sleeping Beauty) of theTc1/mariner superfamily of transposonsfor transgene stabilization2.

During the past decade, substantial efforthas been focused on overcoming severalobstacles that had hampered the clinical appli-cation of gene therapy. Work has focused onthe choice of appropriate disease models andsuitable gene-delivery vehicles for particulartargets and indications. In vector develop-ment, progress has been made with variousvectors in modifying virus tropism, increasingtransgene capacity, enhancing stability andcontrol of transgene expression, and down-modulating host immune responses.

Achieving long-term gene expressionusing gene-deleted adenoviral vectors isproblematic, because the system lacks inte-gration machinery. As a result, several inves-tigators are trying to improve adenoviral

Gene therapy gets the Beauty treatmentGene-deleted adenovirus vectors modified to carry the SleepingBeauty transposon machinery achieve long-term transgenestabilization and expression.R. Michael Linden

R. Michael Linden is an associate professor atthe Carl C. Icahn Institute for Gene Therapyand Molecular Medicine and the Departmentof Microbiology, Mount Sinai School ofMedicine, New York, NY 10029. He is fundedby the National Institutes of Health and byPrimeBioTech, Paris, France(Michael.Linden@mssm.edu).

integration frequencies using differenthybrid vectors. This led Kay’s group to inves-tigate whether they could exploit thepromiscuous integration capabilities of theSleeping Beauty transposon in adenoviral-mediated gene delivery.

Sleeping Beauty, recently awakened fromits evolutionary sleep within the fishgenome3, is recognized for its broad poten-tial in such applications as large-scalemutagenesis, transgenesis, and gene thera-py. Transposable elements of this type arerather promiscuous and have been used astransgene vehicles in plants, bacteria,fungi, insects, and vertebrates. Apparently,Sleeping Beauty requires no host factorsand is therefore competent for transposi-tion in a wide variety of cell types—a char-acteristic that makes it quite amenable forhuman gene transfer purposes.

In the present report, Kay and coworkersdescribe the generation of a gene-deletedadenovirus vector to which the SleepingBeauty transposon machinery has beenadded. The goal of this modification is tomediate transgene integration into the hostgenome after virus-mediated DNA deliveryto liver cells. One problem that needed tobe addressed during the development ofthis system was the inefficiency of trans-gene transfer from the vector to the hostgenome in the presence of Sleeping Beauty.

On the basis of previous findings con-cerning Tc1 transposons, the investigatorsposited that transposition from a circularsupercoiled donor molecule might be moreefficient than transposition from the linearadenoviral form. Accordingly, the investiga-tors modified their vector by flanking theadenovirus-encoded transposons with a Flprecombinase recognition target sequences(FRT)4,5. Upon expression of the Flp recom-binase, the transgene-carrying transposonis excised and circularized, thereby generat-ing the putatively improved substrate forthe Sleeping Beauty transposase (Fig. 1).

Kay’s team then tested the efficiency of thesystem for gene transfer in vivo. They investi-gated whether Sleeping Beauty could functionin the context of the adenovirus–transposonvectors by employing a plasmid recoverystrategy that allowed bacterial recovery oftransposon sequences isolated from injectedliver tissue. In support of the hypothesis, asub-fraction of plasmids (8 of 34) isolatedfrom animals that had received the transpo-son vector as well as the Sleeping Beauty andFlp recombinases contained mouse genomicsequences indicative of transposase-mediatedintegration. Sequence analysis of the transposon–host junctions revealed that thetransposon was indeed flanked by the charac-teristic TA dinucleotides observed in transpo-son integration.

Any two-hybrid-like system that has beendesigned to detect protein–protein interactionscan be converted to a light-based control sys-tem. For example, it should be simple to buildlight-activatable enzymes. Systems that usedimerization-dependent reconstitution of β-galactosidase4,β-lactamase5, or mouse dihydro-folate reductase6 may be made light-activatableby substituting N-terminal phytochrome B andPIF3 for the protein domains that drive dimer-ization. This is a special case of light-forceddimerization. Several systems that can forcedimerization have been previously described7;these use chemicals as non-covalent cross-linking agents to effect dimerization. They arenot as widely used as they perhaps might bebecause of problems with drug toxicity, speci-ficity, permeability, or reversibility. Forceddimerization by light-switchable N-terminalphytochrome B–PIF3 should be a reasonablealternative for the chemical-based systems.

Since it will be possible to force two or moreproteins, from transcription factor subunits toion channel subunits to interact in a light-dependent way, many new approaches will bepossible. For example, it should be possible insome cases to determine dissociation rates invivo by forcing dimerization of, for example, akinase and its protein substrate using N-

terminal phytochrome B and PIF3, illuminat-ing with far-red light to unbind the complex,and then measuring the resulting dissociationrate using fluorescence resonance energy trans-fer. One particularly powerful approach will beto place specific affinity reagents, such as artifi-cial antibodies or random protein aptamers,under light control. It might be possible toconstruct a protein aptamer whose specificbinding region is a composite of two proteinsubunits bought together by light-based dimer-ization. As many biological control mecha-nisms either use, or are dependent upon, pro-tein heteromerization, this report would seemto signal the dawn of the age of light-based bio-logical engineering. Let the revolution begin.

1. Shimizu-Sato, S. et al. Nat. Biotechnol. 20,1041–1044 (2002).

2. Gambetta, G.A & Lagarias, J.C. Proc. Natl. Acad. Sci.USA. 11, 10566–10571 (2001).

3. Gardner, T.S., Cantor, C.R. & Collins, J.J.Construction of a genetic toggle switch inEscherichia coli. Nature 403, 339–342 (2000).

4. Rossi, F.M., Blakely, B.T., Charlton, C.A. & Blau, H.M.Methods Enzymol. 328, 231–251 (2001).

5. Galarneau, A., Primeau, M., Trudeau, L.E. &Michnick, S.W. Nat Biotechnol. 20, 619–622 (2002).

6. Subramaniam, R., Desveaux, D., Spickler, C.,Michnick, S.W., & Brisson, N. Nat. Biotechnol. 19,769–772 (2001).

7. Spencer, D.M., Wandless, T.J., Schreiber, S.L. &Crabtree, G.R. Science. 262, 1019–1024 (1993).

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

reb

iote

chn

olo

gy

The investigators made asecond, important observa-tion upon carrying outBLAST searches of thegenomic sequences theyobtained from the plasmidrecovery approach: as expect-ed, the integration events hadoccurred in different mousechromosomes, indicating arandom selection of chromo-somal acceptor sites. Takentogether, these data indicatethat Sleeping Beauty in com-bination with Flp recombi-nase can stably integrate ade-novirus-encoded transgenesin a random fashion in vivo.Kay and colleagues then tookthe system one step furtherby demonstrating that trans-posase-mediated integrationdoes lead to a stabilization offactor IX expression in mice.

The approach used by Kayand colleagues focuses on thepersistence of therapeutictransgene expression. To provethis principle, the investigators use helper-dependent (gene-deleted) adenoviruses todeliver the transgene-expressing transposons,together with the necessary recombinases, tothe livers of mice. To facilitate an enrichmentfor transgene cassettes that were integratedinto the host genome, Kay’s team performedpartial hepatectomy followed by carbon tetra-chloride injection. These treatments result inthe proliferation of hepatocytes and thus theloss of extrachromosomal vector DNA.Although undoubtedly this system is notmeant for human therapy, the authors ele-gantly use their approach to demonstrate effi-cacy of the Flt/Sleeping Beauty system forgenome integration of transgenes.

Yet several issues remain to be addressedwith regard to the future applicability ofthis approach, especially in light of possiblealternatives for long-term gene expression.The safety of expression of recombinases inhuman cells needs further evaluation.Aside from the possible complications aris-ing from immune responses against thesenonhuman proteins, it is easy to imaginescenarios in which the expression of theserecombination enzymes could lead tounwanted cellular responses.

Because of their efficient random integra-tion, transposon-based systems have beenused for large-scale mutagenesis in variousspecies. Gene delivery using Sleeping Beauty isconceptually no different from these otherstrategies designed to efficiently generate ran-dom insertional mutations. This approachtherefore requires careful safety evaluation if

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

NEWS AND VIEWS

988

it is to be used as a therapy. However, the stabi-lization of therapeutic transgenes that aredelivered by either viral or non-viral vehiclesremains a challenge. With the exception ofadeno-associated virus–based vectors6, nogene-delivery system has been shown to retainthe transgene cassettes over long periods oftime in an extrachromosomal state. Thus,approaches that facilitate safe and efficientintegration need to be evaluated further.Optimally, integration into the humangenome would occur in a targeted, site-specif-ic manner to avoid insertional mutagenesis.The life cycle of the wild-type form of adeno-associated virus suggests that such targetedDNA integration might be possible7.

In conclusion, further development andevaluation of efficient integration systems,together with additional components toprovide a targeting capacity to the recombi-nation mechanism, might overcome thechallenge of safe transgene stabilization.Strategies such as the one presented by Kayand colleagues may provide novel tools forgene delivery and perhaps give new mean-ing to the phrase “everlasting beauty.”

1. Yant, S.R. et al. Nat. Biotechnol. 20, 999–1005.2. Yant, S.R. et al. Nat. Genet. 25, 35–41 (2000).3. Ivics, Z., Hackett, P.B., Plasterk, R.H. & Izsvak, Z.

Cell 91, 501–510 (1997).4. Ng, P., Beauchamp, C., Evelegh, C., Parks, R. &

Graham, F.L. Mol. Ther. 3, 809–815 (2001).5. Rodriguez, C.I. et al. Nat. Genet. 25, 139–140

(2000).6. Monahan, P.E. & Samulski, R.J. Mol. Med. Today 6,

433–440 (2000).7. Linden, R.M. & Berns, K.I. Contrib Microbiol. 4,

68–84 (2000).

Figure 1. The use of a transgene-expressing Sleeping Beautytransposon together with the necessary recombinases allowsintegration.The adenoviral vector, flanked by inverted terminalrepeats (ITR), contains a Sleeping Beauty transposon (yellowbars indicate transposon terminal repeats). Flanking thetransposon with Flp recognition target sequences (FRT, bluetriangles) and expression of Flp recombinase results incircularization of the Sleeping Beauty substrate. These circulardonor molecules undergo transposition, resulting in insertion ofthe transgene into the host genome.

Adenovirus backbone Transgene

Transgene

I. Excision and circularization of transgene

II. Random integration into the host genome

Flp recombinase

Sleeping beautytransposase

ITR ITR

Transgene

Adenovirus backbone

ITR ITR

©B

ob C

rim

i

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

reb

iote

chn

olo

gy