Delivering the goods: viral and non-viral gene therapy systemsand the inherent limits on cargo DNA and internal sequences

Helen Atkinson • Ronald Chalmers

Received: 2 June 2009 / Accepted: 20 December 2009 / Published online: 19 January 2010

� Springer Science+Business Media B.V. 2010

Abstract Viruses have long been considered to be the

most promising tools for human gene therapy. However,

the initial enthusiasm for the use of viruses has been tar-

nished in the light of potentially fatal side effects. Trans-

posons have a long history of use with bacteria in the

laboratory and are now routinely applied to eukaryotic

model organisms. Transposons show promise for applica-

tions in human genetic modification and should prove a

useful addition to the gene therapy tool kit. Here we review

the use of viruses and the limitations of current approaches

to gene therapy, followed by a more detailed analysis of

transposon length and the physical properties of internal

sequences, which both affect transposition efficiency. As

transposon length increases, transposition decreases: this

phenomenon is known as length-dependence, and has

implications for vector cargo capacity. Disruption of

internal sequences, either via deletion of native DNA or

insertion of exogenous DNA, may reduce or enhance

genetic mobility. These effects may be related to host

factor binding, essential spacer requirements or other

influences yet to be elucidated. Length-dependence is a

complex phenomenon driven not simply by the distance

between the transposon ends, but by host proteins, the

transposase and the properties of the DNA sequences

encoded within the transposon.

Keywords DNA recombination � Transposition �Transposon � Length-dependence � Genetic disease �Human, genome � Gene therapy

Introduction

Gene therapy, for many years, has been championed for the

treatment of inherited recessive conditions such as cystic

fibrosis and Duchenne’s muscular dystrophy. More

recently it has been explored for the treatment of cancers,

tissue trauma and polygenetic conditions such as diabetes,

cardiovascular and neurodegenerative disease. Polygenic

conditions are particularly challenging as they go beyond

replacing a single faulty gene with a functioning copy.

Potential approaches include changing the expression pat-

tern of proteins by up regulating beneficial genes or down

regulating deleterious ones. Gene therapy offers the pros-

pect of delivering a true medical revolution: the ability to

manipulate the genome and correct genetic defects causing

disease.

A successful gene therapy system must perform several

functions. In the simplest case, where isolated cells are

treated in vitro, the genetic material must first be delivered

across the cell membrane, which represents a significant

barrier. Once inside the cell, DNA could in principle be

maintained as an episome. However, it is usually more

desirable to maintain the therapeutic construct integrated in

a chromosome. This ensures faithful replication and seg-

regation during any subsequent cycles of cell division. If

the genetic material is in the form of RNA, an additional

reverse transcription step is required to convert the RNA to

DNA. Currently, viral systems provide the most complete

and efficient set of tools for these purposes. Non-viral

integration systems, such as transposons and the bacterio-

phage integrases are under development as additional tools.

However, the non-viral systems lack mechanisms for

delivery across the cell membrane. To achieve the status of

true gene therapy vectors, transposons and integrases must

first be combined with chemical transfection reagents or

H. Atkinson � R. Chalmers (&)

School of Biomedical Sciences, University of Nottingham,

Queen’s Medical Center, Nottingham NG7 2UH, UK

e-mail: chalmers@nottingham.ac.uk; chalmers@bioch.ox.ac.uk

123

Genetica (2010) 138:485–498

DOI 10.1007/s10709-009-9434-3

packaged into viral capsids to facilitate access to the cell

across the cell membrane.

Gene therapy can be administered in vivo and ex vivo.

The nature of a disease often predetermines which method

of delivery will be most beneficial. In vivo applications are,

of course, the most challenging, and may involve the

injection of the vector into the target tissue or into a blood

vessel close to the target area. In vivo treatments have been

explored for tumors (Adams et al. 2009), as well as the

inherited monogenic diseases like cystic fibrosis (Conese

et al. 2008). Ex vivo treatment requires the harvest of

deficient cells from the patient. The cells are then treated

with the vector and returned to the patient having acquired

their new DNA. The treatment of inherited skin conditions

and non-genetic skin damage is being explored with ex

vivo therapy (Warrick et al. 2008). Successful ex vivo gene

therapy has already been achieved for the immune disease

severe combined immune deficiency (SCID) (Cavazzana-

Calvo et al. 2000).

The progress made so far is to be noted and celebrated.

However, many significant hurdles remain. One of these is

to develop a range of different tools that can be used for

different therapeutic purposes and targets, as required.

Viruses are currently the vector of choice and yet very few

phase II or III clinical trials are being conducted (Edelstein

et al. 2007b). This reflects the need to explore new avenues

and find new routes for efficient gene delivery. Transpo-

son-mediated therapy is one of these avenues.

Apart from delivery across the cell membrane, one of

the greatest barriers to successful gene therapy is cargo

capacity. Optimizing a system to carry sufficient genetic

information to correct a complex or subtle genetic defect is

no easy feat: several avenues have been pursued, with

varying degrees of success. Before considering the effects

of cargo DNA on the performance of transposon systems,

we will attempt to place this in context by briefly

describing the current crop of viral vectors and non-viral

integration systems.

Viral vectors

The primary advantage of using viruses is that they are

highly efficient machines for delivering their genetic cargo

across the cell membrane, which otherwise provides a

significant barrier. They require little modification over-

and-above that required to insert the cargo. This makes

viral vectors very attractive compared to the current non-

viral therapeutic approaches which lack the delivery effi-

ciency and nuclear access that viral vectors can provide

(Niidome and Huang 2002). Nevertheless viral vectors

have major limitations.

Here we present a short overview of the retrovirus and

the adenovirus, the most widely used vectors for gene

therapy applications (Edelstein et al. 2007b), and the

adeno-associated virus (AAV) which has a promising

future. We note that other viruses have been investigated,

including pox virus, herpes simplex virus and vaccinia

virus, but we will not discuss these further.

Retroviruses

Retroviruses are small, enveloped, single stranded RNA

viruses with a typical genome capacity of 7–12 kb (Rob-

bins and Ghivizzani 1998; Schaffer et al. 2008). The cretroviruses used in early gene therapy applications have a

strong preference for dividing cells and this limits their use

to mitotic tissues (Mhashilkar et al. 2001; Young et al.

2006). The breakdown of the nuclear envelope during

mitosis is vital to allow the proviral DNA, synthesized in

the cytoplasm, to gain access to the host cell genome. In

contrast, the lentiviruses, such as HIV have a mechanism

for crossing the nuclear membrane. However, they require

host DNA polymerase to complete the reverse transcription

stage and can not therefore infect quiescent cells in the G0

phase of the cell cycle.

Having gained access to the host genome, retroviruses

integrate their DNA using an integrase enzyme closely

related to the DDE-family of transposases (Claeys Bou-

uaert and Chalmers 2010). Of course, viral gene expression

requires integration into regions of euchromatin. Herein

lies both the greatest advantage and drawback of retroviral

therapy. Integration into a chromosome of the host genome

provides stable long term expression of the transgene in the

host cell and any descendents of that cell. However, inte-

grating new DNA into a chromosome carries an inherent

risk of insertional mutagenesis (Bushman 2007).

This problem was highlighted by the treatment of SCID,

a recessive genetic condition, with a retroviral vector in

1999. SCID patients must live in a sterile environment as

their immune system is non-functional and life expectancy

is 2–3 years from birth. This particular trial targeted one

cause of SCID, the absence of the IL2RG gene that encodes

a receptor necessary for communication between cells of

the immune system.

A c retrovirus was used to treat autologous cells ex vivo

to deliver a functioning copy of the missing gene. The cells

developed the previously missing IL2 receptors and caused

remission of the disease in the nine patients treated (Cav-

azzana-Calvo et al. 2000). The trial was a triumph for gene

therapy, but problems arose when between 31 and

68 months post treatment four of the c retrovirus recipients

developed leukemia. Of the four patients affected, three

achieved remission of the disease with chemotherapy.

486 Genetica (2010) 138:485–498

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It was subsequently discovered that the c retrovirus

vector had integrated into the promoter regions of the

proto-oncogenes LMO2, BMI1 and CCND2, leading to

lymphoproliferative disease. Chromosomal translocations

and tumor suppressor gene deletions were also identified

(Hacein-Bey-Abina et al. 2008). It has since been discov-

ered that the c retrovirus used, and retroviruses in general,

have insertion site preferences including transcriptional

start sequences (Laufs et al. 2004). Even so, integration of

the virus into the same site in 3 out of 9 cases was com-

pletely unexpected, and raises the possibility that the cargo

may have contributed in some way to the targeting of the

site.

This was a huge blow for retroviral gene therapy, espe-

cially for conditions less serious than SCID. SCID sufferers

may be willing to take the trade off, being cured of one life

threatening condition only to acquire another, but patients

with less serious conditions may be unwilling to take that

risk. Even though the year 2000 trial has proven retroviruses

to be competent gene vectors for long term gene expression

and phenotype alteration, the overhanging shadow of its less

than desirable insertion preferences have caused retrovi-

ruses to fall from favor. Since 2004 the application of ret-

roviruses as vectors for clinical gene therapy trials has

fallen (Edelstein et al. 2004, 2007a). However, one sub-

sequent retroviral trial has uncovered no adverse effects in

ten patients followed for an average of 4 years following

treatment (Aiuti et al. 2009). Until we better understand

their insertion site selection process retroviruses may find

limited use because of their oncogenic risks.

Adenoviruses

Adenoviruses are non-enveloped double stranded linear

DNA viruses with a natural tropism for infecting the upper

respiratory tract and ocular tissue. They can deliver up to

8 kb of cargo, but this can be extended to 25 kb or more in

the so-called ‘gutless’ versions from which the viral genes

have been deleted. Adenoviruses have an efficient mech-

anism for nuclear entry. Furthermore, their double stranded

DNA genome is not dependent on host DNA polymerases.

In contrast to the retroviruses, which depend on the host

DNA polymerase to complete the reverse transcription

step, adenoviruses can therefore transform post mitotic

cells, providing the potential application in a range of

neurological diseases.

Adenoviruses provide only transient transduction of

target cells, although after appropriate modification their

cargo may be maintained as an episome (Volpers and

Kochanek 2004). The episome may remain transcription-

ally active for the life of the host cell. However, this may

be only a few weeks, as the cell may be recognized and

destroyed by the host immune system (Young et al. 2006).

Even if the cell escapes destruction, episomal DNA is not

replicated and segregated faithfully in mitosis, so the

therapeutic DNA is diluted and eventually lost in the

daughter cells, resulting in transient gene expression.

Mitotic tissues must therefore receive repeated doses to

maintain a long term therapeutic effect. One promising

strategy to avoid this problem is to use some of the cargo

capacity to encode the integration system of the adeno-

associated virus (Goncalves et al. 2008), which will be

described in more detail below.

The need for repeated dosage is the greatest drawback of

the adenoviruses as it inevitably leads to a strong immune

response. The viral capsid and viral DNA cargo are both

thought to be important in provoking the immediate

immune response (McCaffrey et al. 2008). The endogenous

viral cargo genes retained in the vector are ‘leaky’ and may

provoke a cell mediated immune response secondary to the

initial reaction. A single dose may be tolerated, but a

second or third dose is likely to provoke a large immune

response, leading to destruction of the therapeutic vector

and tissue damage within the patient.

The first adenovirus fatality was identified with the

Gelsinger case in 1999. It occurred in a trail aimed at

treating ornithine transcarbamylase (OTC) deficiency, a

recessive X-linked condition. A male aged 18 years

received the highest dose of the vector in the trial and died

4 days later from multiple organ failure caused by a mas-

sive systemic inflammatory response to the vector (Hollon

2000; Raper et al. 2003). This highlights the problems of

adenovirus therapy as the vector administered to Gelsinger

had already been engineered to be of reduced immunoge-

nicity (Gao et al. 1996; Raper et al. 2003).

There are more than 50 human serotypes of the adeno-

virus (Volpers and Kochanek 2004). An initial dose of

adenoviral vector could be followed by a subsequent dose

of the same therapeutic transgene carried by a different

serotype of the vector, reducing the chance of a large scale

immune response.

An alternative approach would be to use gutless ade-

noviruses from which all the genes have been deleted.

These viruses have reduced immunogenicity, but are more

difficult to produce, requiring helper viral proteins supplied

in trans (Alba et al. 2005). However, once manufactured

the virus functions independently with no further help

needed (Jozkowicz and Dulak 2005). Gutless viruses may

still elicit a reduced immune response due to their capsid,

however this may be controllable using immunosuppres-

sants (Roy-Chowdhury and Horwitz 2002).

Adeno-associated virus

Adeno-associated virus (AAV) is a non-pathogenic single

stranded DNA virus with a genome of about 4.7 kb (Young

Genetica (2010) 138:485–498 487

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et al. 2006). Like the related adenoviruses they can infect

dividing or quiescent cells. However, they have a unique

life cycle consisting of a latent phase and a replicative

phase. The replicative phase requires co-infection with an

adenovirus that acts as a helper. During the latent phase

AAV exists integrated into the host cell genome at a spe-

cific location on chromosome 19. This makes it an attrac-

tive system for gene therapy applications. However, there

are reports of AAV causing chromosomal breakage

resulting in rearrangements and deletions (Buning et al.

2008).

AAV has a limited cargo capacity of \4 kb. One strat-

egy to overcome this limitation is trans-splicing of pro-

teins. For example, a therapeutic transgene is split into two

halves, each marked with an intein protein splicing

sequence, and delivered by co-infection of two separate

AAV constructs. This has been achieved in vivo in mice,

and now opens the future possibility of splitting even larger

transgenes into three or four parts (Li et al. 2008). Another

strategy is to clone the cis-acting AAV recombination site

directly into a circular plasmid, and to supply the necessary

viral proteins in trans. Cargo larger than 25 kb can be

integrated in this way, but the therapeutic DNA must be

delivered by chemical transfection methods with limited

efficiency. The mechanism of site specific AAV integration

is not yet well understood because of its complexity.

Integration requires virally encoded endonuclease and he-

licase activities, together with host transcription and rep-

lication factors. The site of integration in the host genome

is dictated by a specific DNA sequence recognized by viral

proteins. However, there appear to be additional restric-

tions that prevent integration at identical sequences located

elsewhere in the genome. Perhaps these are imposed by

chromatin structure which may restrict access to recom-

bining sites. Targeting to the specific integration site is not

absolute and events do occur elsewhere in the genome.

Interestingly, many of these are at sequences homologous

to the cargo contained within the vector. Vectors carrying

the beta-globin locus control region are reported to target

the homologous chromosomal region with an efficiency of

10% (Wang et al. 2005). It therefore appears that elements

of the AAV system, perhaps the endonuclease in combi-

nation with host factors, stimulate homologous recombi-

nation between the cargo and the host genome.

Non-viral integration systems

Less effort has been devoted to the development of non-

viral integration systems owing to the relatively ineffi-

ciency of the transfection methods used to deliver the

DNA, their small cargo capacity and sometimes inefficient

gene expression (Copeland et al. 2007; Flotte 2007).

However, with viral vectors still failing to meet require-

ments in host immune tolerance, further development of

non viral systems are justified (Liu et al. 2004). A range of

integration technologies are needed to provide a variety of

genetic tools for gene therapy; viruses represent one of

these and non viral systems may also find their way into the

tool box. For completeness, we will briefly consider the

non-viral Cre-loxP recombination system and phage C31

integrase before focusing on the potentials of transposon in

more detail.

Cre-loxP and the tyrosine recombinases

Cre is a tyrosine recombinase derived from bacteriophage

P1 (Gorman and Bullock 2000; Van Duyne 2001). Other

members of this family include bacteriophage lambda in-

tegrase, XerCD that resolves chromosome dimers in bac-

teria and the Flp invertase from bakers yeast. Cre catalyzes

reciprocal recombination between two recombination sites

known as loxP. The reaction has no directionality and the

arrangement of the sites dictates whether the result of the

reaction is integration, excision or inversion (Fig. 1). The

system is highly efficient and has been widely used to

generate conditional knockouts in mice by placing the gene

of interest between loxP sites, using conventional cloning

techniques, followed by expression of the recombinase

from a tissue specific promoter. However, this technology

has several pitfalls, such as promoter leakage and unex-

pected epigenetic effects arising from direct transmission

of the Cre protein in the oocyte (Matthaei 2007).

The human genome does not contain any loxP sites.

However, there are a number of pseudo-loxP sites that

provide targets for the integration of transgenes (Thyaga-

rajan et al. 2000). Further discrimination between sites

could be achieved if the sequence specificity of Cre could

be modified sufficiently. However, progress has been dis-

appointing despite the availability of several crystal

structures to guide efforts. It appears that constraints are

imposed by the palindromic symmetry of the recognition

sequences and sequence requirements in the cross-over

regions where recombination occurs.

Expression of the Cre recombinase in mammalian cells

is also thought to be genotoxic, causing chromosomal

aberrations. Another fundamental problem arises from the

lack of directionality in the recombination reaction. Cre

integration exists in an equilibrium with excision. How-

ever, the excision reaction is favored by entropic forces,

and any therapeutic gene successfully integrated into a host

genome is liable to be excised due to the continued pres-

ence of the recombinase (Mizuguchi et al. 2001). The

related lambda integrase has the useful property of direc-

tionality, catalyzing integration or excision depending on

the sequence of the recombining sites and presence of host

488 Genetica (2010) 138:485–498

123

factors. A directional chimeric-Cre recombinase, incorpo-

rating a small fragment of lambda integrase, has been

created but this is also dependent on bacterial host proteins,

which limit its usefulness in gene therapy applications

(Warren et al. 2008).

Bacteriophage C31 integrase

The bacteriophage (/) C31 integrase is a member of the

serine recombinase family that includes the Tn3 resolvase

and a number of transposases (Kuhstoss and Rao 1991;

Gupta et al. 2007; Rowley and Smith 2008; Rowley et al.

2008) The integrase catalyzes reciprocal recombination

between the attP site on the phage and the attB site in the

chromosome of the bacterial host. Integration is unidirec-

tional and does not depend on host factors, providing an

attractive system for gene therapy applications (Fig. 2).

The reaction is irreversible because excision, between the

newly created attL and attR sites, requires host factors

absent in eukaryotic cells.

There are no attB or attP sites in the human genome.

However, a plasmid containing an attB site will integrate at

pseudo-attP sites in the presence of /C31 integrase

(Chalberg et al. 2006). The efficiency of integration at true

attP and attB sites is much higher than at pseudo-sites and

depends on the degree of sequence divergence. For reit-

erative in vitro genome engineering experiments it is

therefore convenient to integrate a bona fide attP site, using

conventional means, to facilitate subsequent large-scale

manipulations.

Sequence analysis suggests that there may be a thousand

pseudo-attP sites in the human genome (Thyagarajan et al.

2001). This probably explains the genotoxicity of /C31

integrase as a result of recombination reactions between

these pseudo sites causing chromosomal aberrations in

human cells (Ehrhardt et al. 2006; Liu et al. 2006). Not-

withstanding these problems, a /C31 integrase-based sys-

tem has been used to successfully treat human cells grown

on mice for the collagen deficiency disease recessive dys-

trophic epidermolysis bullosa (RDEB) (Ortiz-Urda et al.

2003).

The potentials of transposon integration systems

Transposons fall into two broad classes: the retrotranspo-

sons that have an RNA intermediate and the DNA trans-

posons that transpose directly with DNA intermediates.

Among the DNA transposons, the cut-and-paste elements

are the simplest and have been widely used as laboratory

tools for generating insertional mutations e.g. (Sun et al.

2000; Tang et al. 2002). More recently, the cut-and-paste

Fig. 1 Cre/lox recombination. a The Cre recombinase from bacterio-

phage P1 catalyses reciprocal recombination between two DNA

sequences known as loxP sites. The loxP site is present in the

bacteriophage P1 genome. Recombination between a chromosomal

and a plasmid-bourn loxP site yields an integration product. Recom-

bination is fully reversible, yielding an excision product. Excision is

probably more efficient than integration because the recombining sites

are located in cis. It should be noted that in nature the phage loxP site

may also recombine with a related site, loxB, on the bacterial

chromosome (Hoess et al. 1982). This reaction is less efficient and in

many respects resembles the situation when a plasmid-encoded loxPsite recombines with a pseudo-loxP site in a eukaryotic host. In

genome engineering applications two identical loxP sites are usually

employed. b Recombination between loxP sites in the inverted repeat

configuration causes inversion of the intervening sequence

Fig. 2 Bacteriophage C31 integrase recombination. The UC31

integrase catalyses reciprocal recombination between two DNA

sequences known as attB and attP (‘bacteria attachment site’ and

‘phage attachment site’, respectively). The sequences are related, but

not identical. Reciprocal recombination yields an integration product

flanked by two new sequences, attL and attR. Recombination between

attL and attR requires an additional host factor. This factor is absent

in eukaryotic cells and recombination is therefore unidirectional

Genetica (2010) 138:485–498 489

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transposons have been developed for in vivo and ex vivo

gene therapy applications. They have been successfully

applied in animal models and are moving towards human

trials (Balciunas et al. 2006; Williams 2008).

The retroviral integrases, which share a common struc-

tural fold and molecular mechanism with the cut-and-paste

transposons e.g. (Bischerour and Chalmers 2007, 2009;

Bischerour et al. 2009; Claeys Bouuaert and Chalmers

2010), present a number of serious technical difficulties

including insolubility. Transposases too are often prob-

lematic, but there are large numbers to choose from and

promising candidates have been identified. The most

desirable characteristics are solubility, high efficiency and

fidelity. Inevitably no single candidate wins through in all

categories. The mariner family elements are perhaps the

most promising eukaryotic candidates as they are reason-

ably tractable to manipulation in vitro e.g. (Lipkow et al.

2004a, b; Liu et al. 2007; Miskey et al. 2007; Munoz-

Lopez et al. 2008; Claeys Bouuaert and Chalmers 2009).

In gene therapy applications transposons have so far

avoided the problems of host immune response and the

genotoxic effects observed for the viral vectors and the site

specific recombinases, respectively, (Palazzoli et al. 2008).

However, transposons are delivered by transfection meth-

ods that currently lack the efficiency of viral vectors.

Transfection methods are improving every year and may

soon match the efficiency of viruses in some tissues. Tar-

geting of insertions to promoter regions seems to be less of

a problem for transposons, with some preferentially

inserting into non-coding DNA and sites distant from

actively transcribed genes (Williams 2008). Of course, this

may also prove disadvantageous if transgenes are inte-

grated into silenced heterochromatin areas. Nevertheless, it

appears that this is not a great problem for the DNA

transposons which are far less likely than retrotransposons

to target heterochromatic regions.

The problems of insertional mutagenesis and positional

effects on transgene expression could be avoided if trans-

position was targeted to specific sites or even chromosomal

regions. One approach is to fuse a targeting domain to the

transposase, for example, by adding a zinc finger DNA-

binding domain or transcription factor (Maragathavally

et al. 2006; Ivics et al. 2007; Claeys Bouuaert and Chal-

mers 2010). This is feasible as many transposases are

modular, comprising two or more separate functional

domains, and are known to tolerate fusion of additional

domains on the N- or C-terminus.

The vectorization of transposons and integrase-based

systems

In contrast to viruses, transposons and the bacteriophage

integrases lack direct mechanisms to gain entry to the cell

across the membrane. However, synthetic vectors have

been created by combining these systems with chemical

transfection reagents or by packaging in viral capsids.

Chemical transfection reagents are improving year-on-year

and, under ideal conditions, now rival the efficiency of

retrovirus vectors, achieving almost 100% efficiency for

some types of cultured cells e.g. (Montier et al. 2008;

Labas et al. 2010).

The most frequently used strategy is to co-transfect a

recombinase expression plasmid, together with a second

plasmid encoding the therapeutic gene flanked by the

appropriate recombination signals e.g. (Izsvak et al. 2009).

This has proven successful with cultured human cells,

providing long-term high-level expression of the transgene.

However, there remains the unresolved issue of potential

toxicity if lipid- or polymer-based transfection reagents

were to be transfused into patients.

An alternative approach to the use of transfection

reagents is the direct injection of naked DNA into tumor

tissue. This technique has proven successful for introduc-

ing simple protein-expression plasmids and has even been

successfully adapted using high pressure jet technology for

treating tumors (Walther et al. 2008, 2009). Presumably it

is only a matter of time before the high pressure jet and the

transposon technology are combined to provide a trans-

fection-reagent-free method for inducing stable long-term

expression of transgenes.

Transposons and phage integrases have also been vec-

torized by combining them with viruses. When cultured

cells are infected with a helper virus, or are otherwise

engineered to express viral genes, they can be used to

package an artificial viral genome into a viral capsid. The

artificial genomes may be designed to incorporate a

recombinase expression cassette and the transgene of

interest next to the cognate recombination signals. The

viral functions provide efficient entry into the cell, where

expression of the recombinase induces integration of the

transgene. Such ‘‘hybrid vectors’’ have been created, for

example, by packaging the Sleeping Beauty transposon

into herpes simplex virus, adenovirus and lentivirus capsids

(Yant et al. 2002; Izsvak et al. 2009), or packaging the /C31 integrase into an adenovirus capsid (Ehrhardt et al.

2007). Of course, hybrid vectors are subject to many of the

drawbacks affecting standard viral vectors, such as

immunogenicity.

The minimal transposon and the length-dependence

of transposition

Many transposons have a simple structure. They encode a

single transposase gene flanked by inverted terminal

repeats (ITRs) that define the ends of the element. After

binding to the ITRs, and bringing them together in a

490 Genetica (2010) 138:485–498

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synaptic complex, the transposase catalyzes excision of the

element and integration at a new target site (Fig. 3). The

simplicity of the components lends itself to experimental

manipulation. If the transposase is supplied from an

external source, any desired DNA can, in principle, be

mobilized if placed between a pair of ITRs. However, it is

also clear that the sequences between the ITRs can have a

profound effect on the efficiency of transposition. This has

obvious implications for gene therapy applications, and we

must consider not only the properties of the cargo placed

between the ITRs, but also the potentially detrimental

effect of deleting some or all of the native internal

sequences.

While the cargo capacity of viral vectors is limited by

the physical space available in the viral capsid, the amount

of DNA a transposon can carry is in principal unlimited.

However, in practice, when transposon length is increased,

for example by the addition of a therapeutic transgene, its

ability to transpose is reduced. This phenomenon is known

as ‘length-dependence’.

The length-dependence of transposition

Length-dependence was first documented in vivo for the

bacterial transposons IS1 and Tn10/IS10. For IS1, a simple

exponential decrease in transposition of 50% was observed

for each extra kb of DNA added. (Fig. 4a) (Chandler et al.

1982). Similar findings were reported for Tn10/IS10 where

each extra kb caused a *40% reduction in transposition

efficiency (Morisato et al. 1983). These experiments were

performed with chromosomally encoded transposons

where the reduction in transposition can be interpreted as

the result of the increased distance between the ends, and

the decreased probability of achieving the synapsis

required for activity. However, a length penalty of only

15% per extra kb was recorded with Tn10/IS10 elements

encoded on a plasmid (Way and Kleckner 1985). One

factor that may account for this difference is that the

maximum distance between the transposon ends is dictated

by the size of the plasmid backbone, and does not increase

beyond this point as the length of the transposon is

increased.

It seems likely that the length-dependence of chromo-

somally encoded transposons is explained at least in part by

the less efficient synapsis of the transposon ends as they are

placed further apart. The length-dependence of plasmid-

encoded transposons requires a different explanation. We

propose that this is due to suicidal auto-integration that

takes place when the transposase selects an intra-molecular

target site within the element itself. Autointegration has

been detected in many transposons and viruses, but has not

been investigated systematically because it is difficult to

quantify e.g. (Claeys Bouuaert and Chalmers 2009, 2010).

However, autointegration is certainly efficient and diverts

transposition away from productive inter-molecular events.

Experiments with plasmid-encoded copies of the

eukaryotic mariner family transposon Himar1 recorded a

reduction in transposition of about 40% per kb of addi-

tional cargo (Fig. 4b) (Lampe et al. 1998). It therefore

seems likely that the length-dependence of eukaryotic

transposons is similar to their relatives in bacteria, as will

be discussed in more detail below.

Complex length-dependence

The Sleeping Beauty (SB) transposon is a member of the

Tc1/mariner superfamily that was reconstructed from

inactive elements in fish (Ivics et al. 1997). SB has been the

subject of much interest as it was the first DNA transposon

to be reconstituted in vertebrate cells.

Simple length-dependence in the SB system has been

observed in HeLa cells (Fig. 4b). The unmodified SB

transposon is *1.7 kb long and every extra 1 kb added

decreases transposition by *30% (Izsvak et al. 2000).

Similar findings were reported in mouse cells, when effi-

cient transposition was reported with SB elements up to

5.6 kb in length, followed by a loss of transposition activity

with elements greater than 9.1 kb (Karsi et al. 2001). The

results of a third study with SB, performed in HeLa cells,

hinted at a slightly more complex relationship (Geurts et al.

2003). For elements ranging from 2 to 7 kb length-

dependence was simple, though less severe than in the first

Fig. 3 Cut-and-paste transposition. Cut-and-paste transposons are

flanked by inverted repeats. Transposase binds to these sites, brings

them together in a synapsis and cleaves the transposon from the donor

site. The excised transposon is then free to diffuse away from the

donor backbone. Target interactions are established, followed by

integration

Genetica (2010) 138:485–498 491

123

report (Fig. 4c). However, with the 11 kb element there

was a suggestion that length-dependence may have

plateaued. If this plateau is real, it suggests that SB might

accommodate much larger transgenes.

As noted above, simple length-dependence in chromo-

somally encoded copies of Tn10 caused 40% reduction in

transposition per kb of DNA added. However, it was later

discovered that insertions or deletions at the SalI, EcoRI or

Kpn1 endonuclease sites in plasmid-encoded elements did

not always increase or decrease transposition efficiency as

expected (Way and Kleckner 1985). The authors suggest

that transposition is regulated by intrinsic factors within the

element. If so, it seems that the SalI site is an area that can

be compromised by insertion of exogenous DNA, without

impairing transposition efficiency as much as might be

expected. This also implies that other internal sequences

may not be so expendable. The intrinsic properties of

transposon DNA, over-and-above its coding potential, may

therefore affect length-dependence, and may provide ways

to manipulate transposition. Overall, it seems that trans-

position efficiency is dictated by factors in addition to the

distance between the ITRs, and that deleting internal

sequences to make way for transgenes may produce

unexpected results and may not always provide for the

most efficient tools.

Extreme length-dependence at short distances

Most transposon sequences in eukaryotic genomes are

inactive. These may be the evolutionary relics of once-

active elements, or the non-autonomous parasites of active

master-elements that provide transposase activity. Minia-

ture inverted-repeat transposable-elements (MITES) are

non-autonomous transposons that resemble deletion

derivatives of the master element. They are common in

eukaryotes, particularly in plants, but are relatively rare in

bacteria (Buisine et al. 2002; Feschotte et al. 2002; Delihas

2008). The large numbers of MITES in eukaryotic gen-

omes suggests that shorter elements enjoy an advantage

over their full length progenitors. A typical example is

given by the Hsmar1 transposon in the human genome,

where 200 copies of the full length element are accompa-

nied by several thousand copies of the corresponding MITE

(Robertson and Zumpano 1997; Cordaux et al. 2006; Liu

Fig. 4 The length-dependence of transposition. a The length-depen-

dence of IS1 transposition. Transposons were encoded on the bacterial

chromosome and the data was re-plotted from reference (Chandler

et al. 1982). b The length-dependence of the indicated eukaryotic

transposons was re-plotted from references (Izsvak et al. 2000; Geurts

et al. 2003; Balciunas et al. 2006). The transposons were encoded on

bacterial plasmids. Sleeping Beauty (SB) and Tol2 transposition was

measured after transfection of cultured human cells. Himar1 trans-

position reactions were performed in vitro with purified transposase. c

The length-dependence of SB transposition in transfected human cells

was slightly different in independent experiments reported by two

different laboratories. Data was re-plotted from references (Izsvak

et al. 2000; Geurts et al. 2003). d A meta analysis of the transposons

in fish genomes revealed a strong correlation between copy number

and length, which extended to the shortest elements. Short elements

appear to have been amplified compared to their longer progenitors.

Data was re-plotted from reference (Tafalla et al. 2006)

492 Genetica (2010) 138:485–498

123

et al. 2007). A more extensive survey of transposons in fish

showed an exponential relationship between copy number

and length that extends to the shortest elements (Tafalla

et al. 2006) (Fig. 4d). This suggests that there is a dividend

available if the factors responsible for length-dependence

can be identified.

Reduced length-dependence in some elements

The PiggyBac (PB) transposon was isolated from the

genome of the cabbage looper moth and is 2.5 kb long

(Wilson et al. 2007). PB is capable of carrying 9.1 kb of

exogenous DNA, reaching a total length of 14.3 kb, with-

out a severe reduction in transposition efficiency (Ding

et al. 2005). Unfortunately PB seems to favor transcription

start sites as targets for integration, raising concerns about

its suitability for gene therapy applications (Palazzoli et al.

2008).

If increased distance between the ends of a transposon

causes a decrease in transposition, one might expect that

transposons significantly larger than the mariner elements

would have lower transposition efficiencies. The medaka

fish transposon Tol2 is 4.7 kb in length, significantly longer

than the 1.7 kb of SB (Koga and Hori 2001; Kawakami

2007). Tol2 might therefore be expected to have a reduced

transposition frequency in its wild-type form compared to

SB, and that addition of cargo DNA might have a further

detrimental effect on the efficiency of transposition.

There is evidence to suggest that this is not so (Balci-

unas et al. 2006; Hackett 2007). In HeLa cells it appears

that a 5 kb Tol2 element may have the same transposition

efficiency as a 2 kb SB element. Furthermore, Tol2 length-

dependence appears to be minimal and a transposon

exceeding 10 kb in length transposed almost as efficiently

as an element half its size. Since the Tol2 element is nat-

urally longer than most cut-and-paste transposons, it may

have some innate property that helps minimize the loss of

efficiency normally associated with increasing length. Of

course, these experiments are difficult to interpret because

it is uncertain whether the different transposons have been

sufficiently optimized in the different host cell types to

allow for strict comparison. Nevertheless, Tol2 appears to

be an efficient vehicle for long transgenes. Perhaps we

should turn our attention to transposons found at greater

lengths in nature for use as gene delivery vehicles.

Tol1 is particularly interesting in this respect since

examples as long as 18–20 kb have been documented

(Koga et al. 2007). If Tol1 exists in its natural host at such a

length, it may hold promise for artificial manipulation as a

gene vector able to carry large transgene constructs.

Indeed, a 22 kb Tol1 element has already been shown to

transpose, albeit at a reduced efficiency (Koga et al. 2007).

The fact that cargo of this length can be tolerated at some

level holds promise for the future of transposon mediated

gene therapy.

Interestingly, the shortest Tol2 transposon tested, with

only 175 bp of cargo, had the greatest reduction in trans-

position efficiency (Urasaki et al. 2006). This suggests that

a minimal spacer region is needed between the two ends of

the transposon.

The causes and cures for length-dependence

Why are some elements able to accept more cargo than

others, and what is the root cause of length-dependence?

These questions remain largely unanswered. As more cargo

is added, the ITRs of the transposon are moved further

apart and may become more difficult for the transposase to

locate or to maintain in a synapsis once they have been

bound. This was a reasonable interpretation of experiments

in which the transposons are integrated into the bacterial

chromosome. However, as pointed out above, this expla-

nation is not sufficient when the transposons are encoded

on plasmids, which is the case for most of the experiments

with the eukaryotic elements and all of the experiments

performed in vitro. Our favored explanation is that the

probability of suicidal autointegration increases as the

length of the transposon increases. There is also the risk

that the exogenous cargo may contain an insertion site

hotspot which will encourage autointegration.

One successful method for increasing the efficiency of

SB transposition is the so-called ‘sandwich vector’ (Zayed

et al. 2004). In this system the cargo DNA is sandwiched

between two arrays of transposase binding sites that each

resemble miniature SB transposons. The innermost set of

binding sites harbor mutations that prevent cleavage, but

they remain proficient for transposase binding. This

arrangement achieves a threefold increase in efficiency

compared to an equivalent wild type transposon. The

mechanism responsible remains unclear. However, it

appears that the ends of the transposon may be held more

tightly together by the additional transposase binding sites,

possibly increasing the stability of the transpososome.

Unfortunately, the sandwich vector approach may not be

universally applicable as similar constructs with Mos1

failed to provide an increase in transposition in vitro

(Marian Takac and RC, unpublished).

Internal sequences

The nested arrangement of binding sites in the sandwich

vector is reminiscent of the complex sub-terminal sequences

present in many natural transposons. Wild type SB itself has

a bipartite arrangement of binding sites at each end. In other

transposons, such as Mos1, the subterminal sequences,

Genetica (2010) 138:485–498 493

123

although clearly complex, are more difficult to align and

interpret because the internal symmetries are shorter and

more diverse (Auge-Gouillou et al. 2001; Bigot et al. 2005;

Brillet et al. 2007; Sinzelle et al. 2008). Nevertheless, these

internal sequences affect the efficiency of transposition.

Artificial transposons are often constructed using only the

20–30 bp inverted repeats that flank the natural transposon.

However, more efficient vectors can usually be achieved by

including subterminal sequences. Thus, much of the work on

the mechanism of Tn10, for example, was performed with

artificial transposons incorporating hundreds of bp of inter-

nal sequence (Chalmers et al. 1998). The precise distances

over which these enhancements operate are difficult to

quantify, and the mechanism remains largely obscure, not

least because more than one mechanism is likely to be

operating.

Some transposons, such as Tn10 or the Drosophila P

element, for example, are activated by specific host factors

binding to the terminal or subterminal regions (Kaufman

et al. 1989; Chalmers et al. 2000; Crellin and Chalmers

2001). These host factors interact with specific sites near

the transposon end and may help to initiate the reaction, or

modulate the outcome, such as the choice of integration

target site (Sewitz et al. 2003; Crellin et al. 2004; Liu et al.

2005). In other cases, host factors such as the eukaryotic

HMG and bacterial HU and H-NS proteins, may target

distorted DNA structures near the transposon end (Chal-

mers et al. 1998; Zayed et al. 2003; Wardle et al. 2005;

Singh et al. 2008; Whitfield et al. 2009). These distortions

probably arise from the skewed base composition and the

repetition of simple sequence motifs at or near transposon

ends. Distorted DNA probably also promotes transposase

binding directly because the subterminal sequences

enhance transposition in vitro when no other proteins are

present.

Mos1 and peach internal sequences

Mos1 internal sequences have a strong influence on trans-

position (Lohe and Hartl 1996; Hartl et al. 1997; Lozovsky

et al. 2002). In a chromosomal excision assay in Drosophila,

the addition of DNA fragments ranging between 4.5 and

11.9 kb decreased the efficiency of Mos1 transposition.

However, this is not a case of simple length-dependence as

insertions at the SacI restriction endonuclease site were

particularly detrimental. This site is near the center of the

element, about 700 bp from the ITRs, within the transpos-

ase coding region. This effect was probably not due to the

disruption of transposase expression as this protein was

provided in trans in these experiments. It is possible that the

SacI region, which is well conserved, may contain a

transpositional enhancer. Internal sequences may also

function at the single nucleotide level; mutations at bp 993,

and a double mutation at bp 161 and bp 179 reduced

transposition significantly.

Further investigations revealed that the effects of the

internal sequences were not constant. The effects of one

sequence were dependent on which other sequences were

present, on the host species and tissue, and on the type of

assay employed. For example, in a bacterial assay an

artificial Mos1 transposon flanked by two copies of the 30-ITR were more active than the wild type transposon, which

has imperfect 50- and 30- ITRs (Pledger et al. 2004; Pledger

and Coates 2005). When transposition was performed in

mosquito larvae the 30-ITR transposon did not provide

increased activity with the wild type transposase, although

the integrity of the reaction was improved. In contrast, a

mutant transposase did provide increased activity for the

30-ITR transposon in the mosquito.

Mos1 transposition in the bacterial assay was increased

even further by deleting subterminal DNA sequences

adjacent to the ITRs. However, in contrast to the results in

bacteria, and those obtained in vitro using purified com-

ponents, deletion of internal Mos1 sequences was extre-

mely detrimental to transposition in the mosquito (Pledger

et al. 2004; Pledger and Coates 2005).

These results highlight problems with the interpretation

of transgenesis efficiency trials. Even though most cut-and-

paste transposons are independent of host factors, the

internal sequences can have strong positive or negative

effects depending on the environment within the target

cells and the type of assay employed. Strict comparisons

therefore require strict normalization of the experimental

conditions.

Tc3 internal sequences

In Tc3 symmetrical deletion of internal sequences

increased the frequency of transposition (Fischer et al.

1999). The authors concluded that this was due to the

reduction in overall transposon length. While this is a

likely explanation given the pervasive effects of length-

dependence, it is possible that these results should be re-

examined in light of the potential effects of internal

sequences.

PiggyBac internal sequences

PiggyBac (PB) transposition is also dependent on internal

sequences (Li et al. 2001, 2005). Constructs with internal

deletions were active in inter-plasmid embryo assays but

were not able to transform Drosophila with the same effi-

ciency as the full length vector, or PB vectors with less

extensive internal deletions. The enhancer sequences were

located within 66 bp of the 5’ ITR and 378 bp of the 5’

ITR. Shortening of the transposon beyond these limits was

494 Genetica (2010) 138:485–498

123

detrimental for transposition and the authors raised a fur-

ther note of caution by suggesting that this effect may vary

between cell lines. The mechanism responsible remains

unclear but it is probably mediated by the simple repeat

sequences within these regions.

hAT family, internal sequences

Transposition of Tol2, a member of the hAT superfamily,

is also dependent on subterminal sequences. With intact

ITRs, just 200 bp of internal sequence from the 50 end and

150 bp from the 30 end were required for efficient trans-

position (Urasaki et al. 2006). These subterminal regions

contain asymmetrically distributed 5 bp repeat sequences.

Mutations in these repeats caused a severe reduction in

transposition, but the mechanism responsible remains

unclear. In addition to the ITRs and the subterminal

regions, Tol2 excision requires an additional nonspecific

spacer-sequence between the ends (\276 bp).

Subterminal repeats with similar effects on transposition

have been identified in the Tag1 transposon from Arabid-

opsis and the maize transposon Ac (Chatterjee and Star-

linger 1995; Liu et al. 2001). The 3.3 kb Tag1 transposon,

for example, requires 97 bp at the 3’ end and 76 bp at the

5’ end of the element (Liu et al. 2001). These subterminal

regions, like those of PB and Tol2, contain simple repeti-

tive sequences.

Concluding remarks

The DNA transposons have been one of the most useful

tools in bacterial genetics over the last few decades. More

recently they have achieved widespread application in

eukaryotic systems. Successful mutagenesis systems have

been established, and the efficiency of the transposon-

based gene-delivery vectors is rising steadily. Improve-

ments have come from a number of different innovations

such as the optimization of the ITRs and the identification

of hyperactive transposase mutations.

The length-dependence of transposition limits the use-

fulness of transposons in gene therapy and synthetic biol-

ogy applications. The loss of efficiency with large cargo

DNA seems to be a manifestation of several contributing

factors. It is dictated not only by the amount of DNA but

also by specific nucleotide sequences that seem to exert

both positive and negative effects depending on the identity

of the transposon and the host cell type. Elucidation of the

underlying mechanisms will no doubt aid the development

of more efficient gene-delivery vectors.

The overall efficiency of a transposition reaction and its

length-dependence are closely related metrics: a hyperac-

tive transposase mutation will improve the efficiency of

long and short transposons alike. We should therefore

continue to search for ways of optimizing the overall

efficiency of the reactions. Since it has been estimated that

a transposon able to carry only 6 kb of cargo could deliver

70–80% of the genes in the human genome (Essner et al.

2005), gene therapy applications appear to be within reach.

Acknowledgments This work was funded by a grant from the

European Commission (Project SyntheGeneDelivery, N�018716). We

would like to thank Louis Marsh for re-plotting the graphs in Fig. 4

and Zoltan Ivics for critical comments on the manuscript.

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