untitled


Reviews

Nonviral Gene Delivery with the Sleeping Beauty
Transposon System

Zoltán Ivics
1–3

and Zsuzsanna Izsvák
1,2

Abstract

Effective gene therapy requires robust delivery of therapeutic genes into relevant target cells, long-term gene
expression, and minimal risks of secondary effects. Nonviral gene transfer approaches typically result in only
short-lived transgene expression in primary cells, because of the lack of nuclear maintenance of the vector over
several rounds of cell division. The development of efficient and safe nonviral vectors armed with an integrating
feature would thus greatly facilitate clinical gene therapy studies. The latest generation transposon technology
based on the Sleeping Beauty (SB) transposon may potentially overcome some of these limitations. SB was shown
to provide efficient stable gene transfer and sustained transgene expression in primary cell types, including
human hematopoietic progenitors, mesenchymal stem cells, muscle stem/progenitor cells (myoblasts), induced
pluripotent stem cells, and T cells. These cells are relevant targets for stem cell biology, regenerative medicine,
and gene- and cell-based therapies of complex genetic diseases. Moreover, the first-in-human clinical trial has
been launched to use redirected T cells engineered with SB for gene therapy of B cell lymphoma. We discuss
aspects of cellular delivery of the SB transposon system, transgene expression provided by integrated transposon
vectors, target site selection of the transposon vectors, and potential risks associated with random genomic
insertion.

Introduction

The ability to efficiently deliver foreign genes intocells provides the basis of using gene therapy to cure
genetic diseases. The vast majority of gene delivery systems
currently tested in clinical trials are based on viral vectors.
Because viruses are highly specialized at crossing through
cellular membranes by infection, they are efficient at deliv-
ering nucleic acids to target cell populations. However, some
viral vectors, including those derived from adenoviruses or
adeno-associated viruses, remain largely episomal, requiring
readministration in order to maintain a desired level of
transgene expression over time. However, repeated delivery
can provoke immune responses against vector-encoded pro-
teins (reviewed in Hartman et al., 2008). In contrast, retro-
viruses integrate their therapeutic cargo into the genome,
resulting in long-term transgene expression. A concern with
using retroviral vectors is genotoxicity associated with mu-
tagenic effects elicited by insertion of the vector into or near
genes (Hacein-Bey-Abina et al., 2003, 2008; Baum et al., 2004).
Such risk is especially pronounced with gammaretroviral

vectors based on the murine leukemia virus ( MLV) that
preferentially integrate into transcription start sites (Wu et al.,
2003). Furthermore, HIV-derived lentiviral vectors are po-
tential mutagens due to their biased insertion into transcrip-
tion units (Schroder et al., 2002; Bushman, 2003). Indeed,
adverse events provoked by insertional mutagenesis of MLV-
based vectors and resulting aberrant T cell proliferation have
been observed in clinical trials for gene therapy of X-linked
severe combined immunodeficiency (SCID-X1) (Hacein-Bey-
Abina et al., 2003, 2008; Thrasher et al., 2006). Additional
concerns include the possibility that transcriptional silencing
may compromise expression of the integrated transgene (re-
viewed in Ellis, 2005), and that large transgenes may inhibit
viral reverse transcription and packaging, thereby setting
limitations to vector design for clinical use of retroviral vec-
tors. Even though the second-generation, self-inactivating
(SIN) recombinant retroviral vectors that lack strong enhancer
elements in their long terminal repeats (LTRs) (Schambach
et al., 2006, 2007; Modlich et al., 2009) may be able to address
some of the inadvertent side effects such as insertional onco-
genesis, the high costs associated with the manufacture of

1Max Delbrück Center for Molecular Medicine, Berlin 13125, Germany.
2University of Debrecen, Debrecen 4032, Hungary.
3Paul Ehrlich Institute, Langen 63225, Germany.

HUMAN GENE THERAPY 22:1043–1051 (September 2011)
ª Mary Ann Liebert, Inc.
DOI: 10.1089/hum.2011.143

1043



clinical-grade retroviral vectors as well as regulatory issues
limit their widespread clinical translation. As a result, signif-
icant efforts and developments have been made toward
crafting gene transfer vector systems that are as efficient but
safer than viruses in clinical gene transfer.

Nonviral vector systems generally suffer from inefficient
cellular delivery and limited duration of transgene expres-
sion due to the lack of genomic insertion and resulting
degradation and/or dilution of the vector in transfected cell
populations. Developments of nonviral delivery techniques,
including liposomal formulations, nanoparticles, advanced
electroporation methods such as nucleofection, and cell-
penetrating peptides can significantly enhance the transfer of
nucleic acids into therapeutically relevant cell types, even
in vivo. In postmitotic tissues, nonviral vectors may provide
long-lasting transgene expression; however, in the absence of
long-term nuclear maintenance in dividing cell types such as
stem cells, even efficient introduction of nucleic acids into
cells does not guarantee long-term transgene expression. One
class of nonviral vector systems that could potentially offer
long-term expression in dividing cell types is based on
scaffold/matrix attachment region (S/MAR)-containing
episomal vectors that can promote replication and mainte-
nance in mammalian cells (Hagedorn et al., 2011). Yet an-
other type of therapeutic vector would ideally unite the
advantages of integrating viral vectors (i.e., long-lasting
transgene expression) with those of nonviral delivery sys-
tems (i.e., lower immunogenicity, enhanced safety profile
and reduced costs of manufacture). Transposable elements
(transposons) could potentially offer such an alternative.

The Sleeping Beauty Transposon as a Nonviral Vector
for Gene Therapy

DNA transposons are discrete pieces of DNA with the
ability to change their positions within the genome via a cut-
and-paste mechanism called transposition. In nature, these

elements exist as single units containing the transposase gene
flanked by terminal inverted repeats (TIRs) that carry
transposase-binding sites (Fig. 1A). However, under labora-
tory conditions, it is possible to use transposons as bicom-
ponent systems, in which virtually any DNA sequence of
interest can be placed between the transposon TIRs and
mobilized by trans-supplementing the transposase in the
form of an expression plasmid (Fig. 1B) or mRNA synthe-
sized in vitro. In the transposition process, the transposase
enzyme mediates the excision of the element from its donor
plasmid, followed by reintegration of the transposon into a
chromosomal locus (Fig. 1C). This feature makes transposons
natural and easily controllable DNA delivery vehicles that
can be used as tools for versatile applications, including gene
therapy.

On the basis of transposon fossils that are presumed to
have been active > 10 million years ago in fish genomes, an
ancient transposon was resurrected, and named Sleeping
Beauty (SB) after the famous fairy tale collected by the
Brothers Grimm, because it was literally awakened after a
long evolutionary ‘‘sleep’’ (Ivics et al., 1997). SB was the first
transposon ever shown capable of efficient transposition in
vertebrate cells, thereby enabling new avenues for genetic
engineering in animal model species (reviewed in Ivics et al.,
2009) as well as for human gene therapy (Yant et al., 2000;
Ivics and Izsvak, 2006). As a nonviral alternative to viral
vectors, the potential of the SB system has been thoroughly
probed (Izsvák and Ivics, 2004; Fernando and Fletcher, 2006;
Ivics and Izsvak, 2006; VandenDriessche et al., 2009; Hackett
et al., 2010; Izsvák et al., 2010).

The advantage of SB transposon-based gene delivery is
that it combines the favorable features of viral vectors with
those of naked DNA molecules. Namely, owing to perma-
nent genomic insertion of transgene constructs (Fig. 1C),
transposition-mediated gene delivery can lead to sustained
and efficient transgene expression in preclinical animal
models (Hackett et al., 2010). However, in contrast to viral

FIG. 1. General organiza-
tion and use of transposable
elements as gene vectors.
(A) Autonomous transpos-
able elements consist of ter-
minal inverted repeats
(TIRs; black arrows) that
flank the transposase gene.
(B) Bicomponent transposon
vector system for delivering
transgenes that are main-
tained in plasmids. One
component contains a DNA
of interest between the
transposon TIRs carried by a
plasmid vector, whereas the
other component is a trans-
posase expression plasmid,
in which the black arrow
represents the promoter
driving expression of the
transposase. (C) The trans-
poson carrying a DNA of
interest is excised from the

donor plasmid and is integrated at a chromosomal site by the transposase. Color images available online at
www.liebertonline.com/hum

1044 IVICS AND IZSVÁK



vectors, transposon vectors can be maintained and propa-
gated as plasmid DNA (Fig. 1B and C), which makes them
simple and inexpensive to manufacture, an important con-
sideration for the implementation of future clinical trials.
Further advantages of the SB system include its reduced
immunogenicity (Yant et al., 2000), no strict limitation of the
size of expression cassettes (Zayed et al., 2004), and improved
safety and toxicity profiles (Ivics et al., 2007; Moldt et al.,
2007; Walisko et al., 2008; VandenDriessche et al., 2009).
Because transposition proceeds through a cut-and-paste
mechanism that involves only DNA, transposon vectors are
not prone to incorporating mutations by reverse transcrip-
tion (that are generated in retroviral stocks at reasonable
frequencies), and can tolerate larger and more complex
transgenes. Unlike the LTRs of retroviruses, the TIRs of SB
vectors have low enhancer/promoter activity ( Moldt et al.,
2007; Walisko et al., 2008). The insertion of chromatin
boundary elements (insulators) flanking the transposon-
contained expression cassettes (Fig. 2A) to prevent accidental
trans-activation of cellular promoters further improved the
safety profile of the SB system (Walisko et al., 2008). Further-
more, synthetically produced mRNA can serve as a source of
the transposase, thereby limiting the duration of transposase
expression and lowering the risk of ‘‘rehopping’’ of the already
integrated transposon-based vector (Wilber et al., 2006).
Chromosomal integration of SB transposons is random, which
is potentially mutagenic (see below), but no SB-associated
adverse effects have been observed in preclinical animal
studies (Fernando and Fletcher, 2006; Ivics and Izsvak, 2006;
Hackett et al., 2010; Izsvák et al., 2010). Last, SB transposons
can be harnessed to integrate plasmid-based small hairpin
RNA (shRNA) expression cassettes into chromosomes to ob-
tain stable knockdown cell lines by RNA interference (Fig. 2B)
(Kaufman et al., 2005). Such technologies have been evaluated
as a potential approach to the therapy of acquired immuno-
deficiency syndrome by stable RNA interference with SB
vectors knocking down the CCR5 and CXCR4 cell surface
coreceptors that are required for viral entry as a first step to
confer resistance to HIV (Tamhane and Akkina, 2008).

Delivery of the Sleeping Beauty vector system

In ex vivo gene delivery, the therapeutic gene vector is
introduced into a selected cell population that had been
isolated from the patient, and the treated cells are trans-
planted back into the same patient. Because transposons are
not infectious, it is necessary to combine the plasmid-based

transposon vectors with technologies capable of efficient
delivery of these nonviral vectors into cells. Because the ef-
ficiency of transposition is dependent on the efficiency of
uptake of the introduced plasmids into the cell nuclei, de-
livery is a rate-limiting factor in transposition, and is thus of
paramount importance. In principle, any technology devel-
oped for transferring nucleic acids into cells can be combined
with transposon vectors. Unfortunately, there is no generally
applicable method, and procedures must be established for
each cell type. In difficult-to-transfect cells, including pri-
mary stem cells, delivery of transposon-based vectors can be
significantly facilitated by nucleofection, a procedure based
on electroporation that transfers nucleic acids directly into
the nucleus. Indeed, nucleofection facilitated transposition in
various stem cells including CD34 + hematopoietic progeni-
tors (Hollis et al., 2006; Izsvák et al., 2009; Mates et al., 2009;
Sumiyoshi et al., 2009; Xue et al., 2009), primary T cells
(Huang et al., 2008, 2009; Singh et al., 2008) and human em-
bryonic stem cells (Wilber et al., 2007; Orban et al., 2009).
Importantly, in the context of the hematopoietic system, this
ex vivo gene delivery procedure apparently did not com-
promise the potential of transposon-marked CD34 + cells to
differentiate normally into the erythroid, megakaryocytic,
granulocyte/monocyte/macrophage lineage ( Mates et al.,
2009) as well as into the CD4 + CD8 + T, CD19 + B, CD56 +

CD3 - natural killer (NK), and CD33 + myeloid lineages (Xue
et al., 2009). The robustness and feasibility of this nonviral,
transposon-based procedure may significantly facilitate
clinical realization of ex vivo stem cell therapy for the treat-
ment of hematopoietic disorders and cancer, which has led to
its application in humans (Williams, 2008).

In in vivo gene delivery, the therapeutic gene vector is
directly introduced into an organ, where expression of the
therapeutic gene construct is desired. Complexing naked
DNA with polyethylenimine (PEI) followed by intravenous
injection is one of the most effective methods to deliver
therapeutic genes to the lung (Belur et al., 2007). This ap-
proach resulted in long-term expression of luciferase after
delivery of SB transposon vectors to the lungs of mice (Belur
et al., 2003, 2007). Complexing of transposon vectors with PEI
allowed expression of therapeutic levels (10% of normal) of
the blood coagulation factor VIII (FVIII) as well as pheno-
typic correction of the bleeding disorder in a mouse model of
hemophilia A (L. Liu et al., 2006b). In a rat model of pul-
monary hypertension, systemic administration of an SB
vector harboring an endothelial nitric oxide synthase (eNOS)
gene resulted in inhibition of induced pulmonary

FIG. 2. Expression cassettes de-
livered by Sleeping Beauty trans-
poson vectors. (A) A typical
therapeutic expression cassette
contains a ubiquitous or tissue-
specific enhancer/PolII promoter
that drives expression of a thera-
peutic gene. To enhance the safety
of such a vector, the expression
cassette might be flanked by in-
sulator elements that will block
trans-activation of endogenous
promoters by the transposon insertion, and simultaneously protect the expression of the therapeutic gene from position
effects. (B) Knockdown expression cassette including a PolII promoter that drives expression of a marker gene and a PolIII
promoter that drives expression of a short hairpin RNA (shRNA). Color images available online at www.liebertonline.com/hum

NONVIRAL GENE DELIVERY WITH SB 1045



hypertension (L. Liu et al., 2006a). Furthermore, PEI-based
systemic delivery of the SB transposon system encoding the
human indoleamine-2,3-dioxygenase (hIDO) gene was eval-
uated in the context of lung transplantation-associated
chronic complications, and found to elicit a remarkable
therapeutic response, as evidenced by near normal pulmo-
nary function in lung allografts (H. Liu et al., 2006). Last, SB-
mediated gene transfer significantly increased survival of
mice bearing human glioblastoma xenografts by expressing
antiangiogenic gene products (Ohlfest et al., 2005a), and
improved the efficacy of immunotherapy by facilitating
sustained cytokine expression after local, intratumoral in-
jections of vector–PEI complexes (Wu et al., 2007).

As an alternative to complexing with a chemical com-
pound, the use of physical delivery systems, such as hy-
drodynamic injection, can facilitate cellular uptake of naked
DNA molecules (Liu et al., 1999; Zhang et al., 1999). This
procedure involves rapid injection of a large volume of DNA
solution through the tail vein. Delivery of therapeutic SB
transposon constructs by hydrodynamic injection (Bell et al.,
2007) was first demonstrated to be applicable to mediate
stable, long-term expression in mouse liver by Yant and
colleagues, who reported sustained expression of a1-
antitrypsin in C57BL/6 mice (Yant et al., 2000). This proce-
dure has been successfully applied to confer a therapeutic
benefit in several animal models of human diseases, in-
cluding hemophilia B after delivery of human clotting factor
IX (FIX) as a therapeutic gene product in FIX-deficient mice
(Yant et al., 2000) and tyrosinemia after hydrodynamic de-
livery of a fumarylacetoacetate hydrolase (FAH)-encoding
SB transposon vector into the livers of FAH-deficient mice
that showed selective outgrowth of genetically corrected
hepatocytes ( Montini et al., 2002). Additional, successful
preclinical testing of the SB system has been established in
disease models for hemophilia A (Ohlfest et al., 2005b) and
mucopolysaccharidosis (Aronovich et al., 2007, 2009).

Cell type-specific expression of the transgene is often
desirable in order to exclude or limit ectopic transgene ex-
pression. One way to achieve this is to equip the transpo-
son-contained expression cassette with tissue-specific
promoters; this was shown to confer erythroid-specific ex-
pression of b-globin in a sickle cell disease model (Zhu
et al., 2007). Another strategy for targeted expression of
therapeutic gene constructs is their targeted delivery into
tissues of interest. Hyaluronan- and asialoorosomucoid-
coated nanocapsules, were found to target SB-based vectors
carrying an FVIII transgene to hepatocytes in vivo and to
improve the phenotype of hemophilia A mice, after intra-
venous injection (Kren et al., 2009). Similarly, packaging an
SB vector expressing chUGT1A1 into proteoliposomes in-
corporating a fusogenic glycoprotein that promoted asia-
loglycoprotein receptor (ASGPR)-mediated endocytosis
allowed successful in vivo delivery and sustainable, thera-
peutic gene expression in the hepatocytes of a rat model of
Crigler-Najjar syndrome type 1 (Wang et al., 2009). Im-
portantly, in neither study was a significant host immune
response toward the gene delivery vehicle or the transgene
product observed (Kren et al., 2009; Wang et al., 2009). In
summary, when combined with nonviral delivery ap-
proaches, the SB transposon system shows considerable
efficacy in providing sustained levels of therapeutic gene
expression both ex vivo and in vivo.

Target site selection of Sleeping Beauty
and its experimental manipulation

The insertion pattern of most transposons is nonrandom,
showing characteristic preferences for insertion sites at the
primary DNA sequence level, and ‘‘hotspots’’ and ‘‘cold re-
gions’’ on a genome-wide scale. SB displays considerable
specificity in target site selection at the primary DNA se-
quence level in that TA dinucleotides are obligate target sites
(Vigdal et al., 2002). A palindromic AT-repeat consensus se-
quence with bendability and hydrogen-bonding potential
was found to constitute the preferred target site (Vigdal et al.,
2002). It was later shown that a characteristic deformation of
the DNA sequence may be a recognition signal for target
selection (Liu et al., 2005). This deformation, and the likeli-
hood that a particular TA will be targeted by SB, can be
computationally predicted (Liu et al., 2005), which may allow
a theoretical assessment of risks associated with transposon
insertions in particular genomic regions (Geurts et al., 2006).
Nevertheless, a systematic assessment of potential genotoxic
effects associated with genomic integration of transposon
vectors will need to be performed either in cell-based assays
and/or in animal models to provide clinically relevant data.

In contrast to the considerable specificity at the primary
DNA sequence level, SB integration can be considered fairly
random at the genomic level (Vigdal et al., 2002; Yant et al.,
2005; Moldt et al., 2011). Roughly one-third of SB insertions
in mouse and human cells occur in transcribed regions, and
because genes cover about one-third of the genome, such
frequency suggests neither preference for nor disfavoring
insertion into genes. The vast majority of those insertions
that occur in genes are located in introns (Vigdal et al., 2002),
and, in contrast to most integrating viral vectors, the tran-
scriptional status of targeted genes apparently does not in-
fluence the integration profile of SB (Yant et al., 2005).

Target site selection properties suggest that SB might be
safer for therapeutic gene delivery than the integrating viral
vectors that are currently used in clinical trials. However,
with any vector that integrates into chromosomes in a nearly
random manner (theoretically, the SB transposon could in-
sert into any of the *108 TA sites in the human genome)
comes the potential risk of insertional mutagenesis leading to
transcriptional activation or inactivation of cellular genes
(Baum et al., 2004). Integration of the vector into a gene or its
regulatory elements can knock out the gene, overexpress the
gene, or alter its spatio/temporal expression pattern. Such
genotoxic effects can have devastating consequences for the
cell and the whole organism, including the development of
cancer, as discussed previously (Baum et al., 2004). As a
possible strategy, introducing an imposed bias into the in-
sertion profile, ideally, targeted integration of the therapeu-
tic gene into a ‘‘safe’’ site in the human genome, could
lower or eliminate possible hazards to the host cell. For tar-
geted transposon insertion at least one component of the
transposon system, either the transposon vector DNA or the
transposase (or factors interacting with either of these com-
ponents), needs to be engineered to be physically linked or
interact with a heterologous DNA-binding domain (DBD),
which is to tether the transposase/transposon complex to
defined sites in the human genome, and to facilitate inte-
gration of the transposon into adjacent DNA (Voigt et al.,
2008). Fusions of the SB transposase with the GAL4 DBD

1046 IVICS AND IZSVÁK



showed an enrichment of transposon insertions in a *400-bp
window around GAL4-binding sites in plasmid targets in
human cells (Yant et al., 2007). Target-selected SB insertion
was also assessed by employing a molecular strategy based
on engineering a LexA operator sequence into an SB trans-
poson vector. Targeted transposition events into chromo-
somal S/MAR sequences as well as a chromosomally
integrated tetracycline response element were recovered by
coexpressing targeting fusion proteins containing LexA and
either the SAF box, a protein domain that binds to S/MARs,
or the tetracycline repressor (TetR) (Ivics et al., 2007). Pre-
sumably, these targeting fusion proteins ‘‘sandwiched’’ the
transposon and target DNA sites, allowing local insertion
events. Last, targeted SB transposition within a 2.5-kb win-
dow around a chromosomally located tetracycline response
element was observed at a frequency of > 10% (Ivics et al.,
2007) by coexpressing the SB transposase with a targeting
fusion protein consisting of TetR and a subdomain of the SB
transposase spanning the N-terminal helix–turn–helix do-
main (N57) that mediates protein–protein interactions be-
tween transposase subunits (Izsvák et al., 2002). Ongoing
work in the authors’ laboratory is dedicated to determining
whether SB transposition can potentially be directed to
physiologically relevant, endogenous sites in the human
genome, by using both naturally occurring as well as syn-
thetic DBDs.

Transgene expression

Any transgene vector system should ideally provide long-
term expression of transgenes. By using classical, plasmid-
based, nonviral delivery approaches, expression from the
extrachromosomal plasmid rapidly declines after delivery.
Transgenes delivered by nonviral approaches often form
long, repeated arrays (concatemers) that are targets for
transcriptional silencing by heterochromatin formation. In
addition, long-term expression of transgenes delivered by
retroviruses has been shown to be compromised by tran-
scriptional silencing ( Jahner et al., 1982). It was shown that
the zinc finger protein ZFP809 bridges the integrated pro-
viral DNA of the murine leukemia virus and the tripartite
motif-containing 28 transcriptional corepressor in embryonic
stem cells (Wolf and Goff, 2009). Thus, sequence elements in
the vector itself can predispose the cargo for silencing. The
cut-and-paste mechanism of DNA transposition results in a
single copy of the transgene per insertion locus, and thus
concatemer-induced gene silencing is unlikely to be an issue
with transposition-mediated gene transfer. Indeed, Gra-
bundzija and colleagues found that transposon insertions
delivered by the SB system only rarely (< 2% of all insertions)
undergo silencing in HeLa cells (Grabundzija et al., 2010).
Furthermore, stable transgene expression observed in *300
independent insertions in this study suggests that SB
rarely targets heterochromatic chromosomal regions for in-
sertion, and that it is unlikely that certain sequence motifs in
the transposon vector are recognized by mediators of si-
lencing in the cell. An additional factor that may provoke
transgene silencing is the cargo DNA, particularly the type of
promoter used to drive expression of the gene of interest.
Indeed, it was previously shown that transgene constructs
delivered into mouse cells by SB transposition can be subject
to epigenetic regulation by CpG methylation and that a de-

terminant of epigenetic modifications of the integrating
transposon vector is the cargo transgene construct, with the
promoter playing a major role (Garrison et al., 2007). How-
ever, with careful promoter choice, several studies have es-
tablished that SB-mediated transposition provides long-term
expression in vivo, as discussed previously. Notably, stable
transgene expression from SB vectors was seen in mice after
gene delivery in the liver (Yant et al., 2000; Ohlfest et al.,
2005b; Aronovich et al., 2009; Kren et al., 2009), lung (Belur
et al., 2003; L. Liu et al., 2006b), brain (Ohlfest et al., 2005a),
and blood after hematopoietic reconstitution in vivo ( Mates
et al., 2009; Xue et al., 2009). Thus, although our under-
standing of all the factors that will ultimately determine the
expressional fate of an integrated transposon is still rudi-
mentary, it appears that transposon vectors have the capacity
to provide long-term expression of transgenes both in vitro
and in vivo.

Gene transfer into stem cells by the SB100X
hyperactive transposase

The hyperactive variant of the SB transposase, SB100X,
yields efficient stable gene transfer after nonviral gene de-
livery into therapeutically relevant primary cell types, in-
cluding stem or progenitor cells. For example, the use of the
SB100X system yielded robust gene transfer efficiencies into
human hematopoietic progenitors ( Mates et al., 2009; Xue
et al., 2009), mesenchymal stem cells, muscle stem/progeni-
tor cells (myoblasts), induced pluripotent stem cells (iPSCs)
(Belay et al., 2010), and T cells ( Jin et al., 2011). These cells are
relevant targets for stem cell biology and for regenerative
medicine and gene- and cell-based therapies of complex ge-
netic diseases. Importantly, expression of the SB100X hy-
peractive transposase did not adversely influence the
differentiation or function of these adult stem/progenitor
cells, nor was there any evidence of any cytogenetic abnor-
malities (Belay et al., 2010). In the context of iPSC technology,
the ability to coax the differentiation of pluripotent stem cells
into clinically relevant, transplantable cell types is a key step
toward their ultimate use in clinical applications, especially
because undifferentiated iPSCs pose an intrinsic tumori-
genic risk (Yamanaka, 2009). It was demonstrated that SB
transposon-mediated delivery of the myogenic PAX3 tran-
scription factor into iPSCs coaxed their differentiation into
MyoD + myogenic progenitors and multinucleated myofibers
(Belay et al., 2010), suggesting that PAX3 may serve as a
myogenic ‘‘molecular switch’’ in iPSCs, a finding that has
implications for cell therapy of congenital degenerative
muscle diseases, including Duchenne muscular dystrophy.
Thus, the SB100X hyperactive transposase holds great
promise for ex vivo and in vivo gene therapies.

Future Considerations/Outlook

There has been steadily growing interest in applying the
SB system for gene therapy (Izsvák and Ivics, 2004; Essner
et al., 2005; Hackett et al., 2005, 2010; Ivics and Izsvak, 2006;
Izsvák et al., 2010) in the context of several conditions in-
cluding hemophilia A and B (Yant et al., 2000; Ohlfest et al.,
2005b; L. Liu et al., 2006b; Kren et al., 2009; Hausl et al., 2010),
junctional epidermolysis bullosa (Ortiz-Urda et al., 2002),
tyrosinemia I ( Montini et al., 2002), Huntington disease
(Chen et al., 2005), sickle cell disease (Zhu et al., 2007),

NONVIRAL GENE DELIVERY WITH SB 1047



mucopolysaccharidosis (Aronovich et al., 2007, 2009), cancer
(Ohlfest et al., 2005a; Peng et al., 2009; Jin et al., 2011), and
type 1 diabetes (He et al., 2004). In addition, important steps
have been made toward SB-mediated gene transfer in the
lung for potential therapy of a1-antitrypsin deficiency, cystic
fibrosis, and a variety of cardiovascular diseases (Belur et al.,
2003; Liu et al., 2004).

The first clinical application of the SB system is currently
ongoing, using autologous T cells gene-modified with SB
vectors (Williams, 2008) carrying a chimeric antigen receptor
(CAR) to render the T cells cytotoxic specifically toward
CD19-positive lymphoid tumors (Huang et al., 2008; Singh
et al., 2008). Lymphocytes represent a suitable initial platform
for testing new gene transfer systems, as T cells can be ge-
netically modified by viral and nonviral approaches without
apparent resulting genotoxicity (Bonini et al., 2003). Ad-
vantages of using the SB system for genetic modification of
T cells include the ease and reduced cost associated with the
manufacturing of clinical-grade, plasmid-based vectors
compared with recombinant viral vectors.

Sleeping Beauty faces a number of challenges before
widespread clinical translation. For in vivo delivery into the
liver, the hydrodynamic technology needs to be calibrated
first to large-animal models and then eventually to humans.
This is not a simple task to solve, but important steps have
been made toward this goal by developing computer-
assisted protocols (Suda et al., 2008). Another complicating
issue with DNA delivery to the liver is the resulting tissue
damage and the associated (at least transiently) elevated
values of liver enzymes that may compromise clinical ap-
plication of these procedures. A major hurdle in ex vivo de-
livery of the transposon components into relevant primary
cell types is the toxicity of the transfection/electroporation
protocols that is typically observed. In situations in which
target cells are scarce and/or culturing and expansion of the
transfected cells are impossible or cannot be solved without
compromising cell identity and grafting potential, cytotox-
icity of the transfection procedures is a serious issue that may
undermine clinical applications. Development of nonviral
chemical reagents promoting plasmid DNA delivery with
reduced toxicity may provide a solution. Alternatively, the
development of hybrid vector systems combining the natural
ability of viruses to traverse cell membranes with efficient
genomic insertion mediated by the SB system is a promising
strategy. Indeed, components of the SB transposon have
been incorporated into integrase-defective lentiviral particles
that showed efficient gene transfer in a range of human cell
types and an insertion profile favorable to conventional
lentiviral vectors (Staunstrup et al., 2009; Vink et al., 2009;
Moldt et al., 2011). Hybrid adenovirus–SB vectors (Yant et al.,
2002) have been used to efficiently deliver SB transposon
vectors expressing FIX into the liver in a hemophilic dog
model (Hausl et al., 2010). Retroviral vectors disabled in
generating a cDNA copy of the retroviral vector have been
shown to deliver the SB transposase mRNA into target cells
with impressive efficiency (Galla et al., 2011). Last, herpes
simplex virus vectors with a tropism toward infecting neural
progenitor cells have been used to target SB insertions in the
central nervous system in an in utero gene delivery system in
the mouse (Bowers et al., 2006; de Silva et al., 2010). Fur-
thermore, not only the procedures that are used to deliver
the transposon vector components into cells, but those

components themselves, may elicit unwanted effects in the
cell. For example, it was found that overexpression of the SB
transposase can have cytotoxic effects (Galla et al., 2011). The
molecular mechanism of transposase cytotoxicity is currently
not understood, but it is unlikely to be due to uncontrolled
cleavage of genomic DNA by the transposase (Galla et al.,
2011). At any rate, careful dosing of the transposase as well
as the transposon donor plasmids in gene delivery experi-
ments appears to be of fundamental importance; luckily,
with plasmid-based vectors such as the SB transposon sys-
tem, this can easily be achieved. Last, potential genotoxic
effects elicited by transcriptional upregulation of proto-
oncogenes and other signaling genes on random transposon
insertion is a relatively unexplored area of research. In-
vestigations into these questions will be required to docu-
ment the safety of the SB system for prospective clinical
trials. The next phase of research will undoubtedly focus on
these issues to introduce the SB transposon system as a
nonviral gene delivery tool for gene therapy.

Acknowledgments

Work in the authors’ laboratories was supported by EU
FP6 (INTHER) and EU FP7 (PERSIST and InduStem), grants
from the Deutsche Forschungsgemeinschaft SPP1230 ‘‘Me-
chanisms of Gene Vector Entry and Persistence,’’ and from
the Bundesministerium für Bildung und Forschung (NGFN-
2, NGFNplus, iGene, InTherGD, and ReGene).

Author Disclosure Statement

No competing financial interests exist.

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Address correspondence to:
Dr. Zoltan Ivics

Max Delbruck Center for Molecular Medicine
Robert Rössle Strasse 10

D-13125 Berlin
Germany

E-mail: zivics@mdc-berlin.de

Received for publication August 5, 2011;
accepted after revision August 22, 2011.

Published online: August 25, 2011.

NONVIRAL GENE DELIVERY WITH SB 1051